Texas A&M University

Graduate Research Assistant

J. Brandon worked at Texas A&M University as a Graduate Research Assistant

Georgia Institute of Technology

Assistant Professor

George W. Woodruff School of Mechanical Engineering
Parker H. Petit Institute for Bioengineering and Bioscience
Wallace H. Coulter Department of Biomedical Engineering

Georgia Institute of Technology

Associate Professor

George W. Woodruff School of Mechanical Engineering
Parker H. Petit Institute for Bioengineering and Bioscience
Wallace H. Coulter Department of Biomedical Engineering

EPFL

Research Scientist

J. Brandon worked at EPFL as a Research Scientist

LymphaTech Inc.

Co-Founder

LymphaTech is working to improve the quality of life of breast cancer survivors by developing innovative technologies that allow for better treatment and prevention of lymphedema, a swelling disease that affects nearly half of all breast cancer survivors.

The relationship between lymphangion length and maximum pressure generation established through in vivo imaging and computational modeling

American Journal of Physiology

The intrinsic contraction of collecting lymphatic vessels serves as a pumping system to propel lymph against hydrostatic pressure gradients as it returns interstitial fluid to the venous circulation. We propose and validate that the maximum opposing outflow pressure along a chain of lymphangions at which flow can be achieved increases with the length of chain. Using minimally invasive near-infrared imaging to measure the effective pumping pressure at various locations in the rat tail, we demonstrate increases in the pumping pressure along the length of the tail. Computational simulations based on a microstructurally-motivated model of a chain of lymphangions informed from biaxial testing of isolated vessels was utilized to provide insight into the pumping mechanisms responsible for the pressure increases observed in vivo. These models suggest that the number of lympangions in the chain and smooth muscle cell force generation play a significant role in determining the maximum outflow pressure, while frequency of contraction has no effect. In vivo administration of nitric oxide (NO) attenuates lymphatic contraction, subsequently lowering the effective pumping pressure. Computational simulations suggest the reduction in contractile strength of smooth muscle cells in the presence of NO can account for the reductions in outflow pressure observed along the lymphangion chain in vivo. Thus combining computation modeling with multiple measurements of lymphatic pumping pressure provides a method for approximating intrinsic lymphatic muscle activity non-invasively in vivo, while also providing insight into factors that determine the extent that a lymphangion chain can transport fluid against an adverse pressure gradient.

The relationship between lymphangion length and maximum pressure generation established through in vivo imaging and computational modeling

American Journal of Physiology

The intrinsic contraction of collecting lymphatic vessels serves as a pumping system to propel lymph against hydrostatic pressure gradients as it returns interstitial fluid to the venous circulation. We propose and validate that the maximum opposing outflow pressure along a chain of lymphangions at which flow can be achieved increases with the length of chain. Using minimally invasive near-infrared imaging to measure the effective pumping pressure at various locations in the rat tail, we demonstrate increases in the pumping pressure along the length of the tail. Computational simulations based on a microstructurally-motivated model of a chain of lymphangions informed from biaxial testing of isolated vessels was utilized to provide insight into the pumping mechanisms responsible for the pressure increases observed in vivo. These models suggest that the number of lympangions in the chain and smooth muscle cell force generation play a significant role in determining the maximum outflow pressure, while frequency of contraction has no effect. In vivo administration of nitric oxide (NO) attenuates lymphatic contraction, subsequently lowering the effective pumping pressure. Computational simulations suggest the reduction in contractile strength of smooth muscle cells in the presence of NO can account for the reductions in outflow pressure observed along the lymphangion chain in vivo. Thus combining computation modeling with multiple measurements of lymphatic pumping pressure provides a method for approximating intrinsic lymphatic muscle activity non-invasively in vivo, while also providing insight into factors that determine the extent that a lymphangion chain can transport fluid against an adverse pressure gradient.

Leukotriene B4 antagonism ameliorates experimental lymphedema

Science Translational Medicine

Acquired lymphedema is a cancer sequela and a global health problem currently lacking pharmacologic therapy. We have previously demonstrated that ketoprofen, an anti-inflammatory agent with dual 5-lipoxygenase and cyclooxygenase inhibitory properties, effectively reverses histopathology in experimental lymphedema. We show that the therapeutic benefit of ketoprofen is specifically attributable to its inhibition of the 5-lipoxygenase metabolite leukotriene B4 (LTB4). LTB4 antagonism reversed edema, improved lymphatic function, and restored lymphatic architecture in the murine tail model of lymphedema. In vitro, LTB4 was functionally bimodal: Lower LTB4 concentrations promoted human lymphatic endothelial cell sprouting and growth, but higher concentrations inhibited lymphangiogenesis and induced apoptosis. During lymphedema progression, lymphatic fluid LTB4 concentrations rose from initial prolymphangiogenic concentrations into an antilymphangiogenic range. LTB4 biosynthesis was similarly elevated in lymphedema patients. Low concentrations of LTB4 stimulated, whereas high concentrations of LTB4 inhibited, vascular endothelial growth factor receptor 3 and Notch pathways in cultured human lymphatic endothelial cells. Lymphatic-specific Notch1−/− mice were refractory to the beneficial effects of LTB4 antagonism, suggesting that LTB4 suppression of Notch signaling is an important mechanism in disease maintenance. In summary, we found that LTB4 was harmful to lymphatic repair at the concentrations observed in established disease. Our findings suggest that LTB4 is a promising drug target for the treatment of acquired lymphedema.

The relationship between lymphangion length and maximum pressure generation established through in vivo imaging and computational modeling

American Journal of Physiology

The intrinsic contraction of collecting lymphatic vessels serves as a pumping system to propel lymph against hydrostatic pressure gradients as it returns interstitial fluid to the venous circulation. We propose and validate that the maximum opposing outflow pressure along a chain of lymphangions at which flow can be achieved increases with the length of chain. Using minimally invasive near-infrared imaging to measure the effective pumping pressure at various locations in the rat tail, we demonstrate increases in the pumping pressure along the length of the tail. Computational simulations based on a microstructurally-motivated model of a chain of lymphangions informed from biaxial testing of isolated vessels was utilized to provide insight into the pumping mechanisms responsible for the pressure increases observed in vivo. These models suggest that the number of lympangions in the chain and smooth muscle cell force generation play a significant role in determining the maximum outflow pressure, while frequency of contraction has no effect. In vivo administration of nitric oxide (NO) attenuates lymphatic contraction, subsequently lowering the effective pumping pressure. Computational simulations suggest the reduction in contractile strength of smooth muscle cells in the presence of NO can account for the reductions in outflow pressure observed along the lymphangion chain in vivo. Thus combining computation modeling with multiple measurements of lymphatic pumping pressure provides a method for approximating intrinsic lymphatic muscle activity non-invasively in vivo, while also providing insight into factors that determine the extent that a lymphangion chain can transport fluid against an adverse pressure gradient.

Leukotriene B4 antagonism ameliorates experimental lymphedema

Science Translational Medicine

Acquired lymphedema is a cancer sequela and a global health problem currently lacking pharmacologic therapy. We have previously demonstrated that ketoprofen, an anti-inflammatory agent with dual 5-lipoxygenase and cyclooxygenase inhibitory properties, effectively reverses histopathology in experimental lymphedema. We show that the therapeutic benefit of ketoprofen is specifically attributable to its inhibition of the 5-lipoxygenase metabolite leukotriene B4 (LTB4). LTB4 antagonism reversed edema, improved lymphatic function, and restored lymphatic architecture in the murine tail model of lymphedema. In vitro, LTB4 was functionally bimodal: Lower LTB4 concentrations promoted human lymphatic endothelial cell sprouting and growth, but higher concentrations inhibited lymphangiogenesis and induced apoptosis. During lymphedema progression, lymphatic fluid LTB4 concentrations rose from initial prolymphangiogenic concentrations into an antilymphangiogenic range. LTB4 biosynthesis was similarly elevated in lymphedema patients. Low concentrations of LTB4 stimulated, whereas high concentrations of LTB4 inhibited, vascular endothelial growth factor receptor 3 and Notch pathways in cultured human lymphatic endothelial cells. Lymphatic-specific Notch1−/− mice were refractory to the beneficial effects of LTB4 antagonism, suggesting that LTB4 suppression of Notch signaling is an important mechanism in disease maintenance. In summary, we found that LTB4 was harmful to lymphatic repair at the concentrations observed in established disease. Our findings suggest that LTB4 is a promising drug target for the treatment of acquired lymphedema.

Post-prandial lymphatic pump function after a high-fat meal: a characterization of contractility, flow, and viscosity

American Journal of Physiology - Gastrointestinal and Liver Physiology

Dietary lipids are transported from the intestine through contractile lymphatics. Chronic lipid loads can adversely affect lymphatic function. However, the acute lymphatic pump response in the mesentery to a postprandial lipid meal has gone unexplored. In this study, we used the rat mesenteric collecting vessel as an in vivo model to quantify the effect of lipoproteins on vessel function. Lipid load was continuously monitored by using the intensity of a fluorescent fatty-acid analog, which we infused along with a fat emulsion through a duodenal cannula. The vessel contractility was simultaneously quantified. We demonstrated for the first time that collecting lymphatic vessels respond to an acute lipid load by reducing pump function. High lipid levels decreased contraction frequency and amplitude. We also showed a strong tonic response through a reduction in the end-diastolic and systolic diameters. We further characterized the changes in flow rate and viscosity and showed that both increase postprandially. In addition, shear-mediated Ca(2+) signaling in lymphatic endothelial cells differed when cultured with lipoproteins. Together these results show that the in vivo response could be both shear and lipid mediated and provide the first evidence that high postprandial lipid has an immediate negative effect on lymphatic function even in the acute setting.

The relationship between lymphangion length and maximum pressure generation established through in vivo imaging and computational modeling

American Journal of Physiology

The intrinsic contraction of collecting lymphatic vessels serves as a pumping system to propel lymph against hydrostatic pressure gradients as it returns interstitial fluid to the venous circulation. We propose and validate that the maximum opposing outflow pressure along a chain of lymphangions at which flow can be achieved increases with the length of chain. Using minimally invasive near-infrared imaging to measure the effective pumping pressure at various locations in the rat tail, we demonstrate increases in the pumping pressure along the length of the tail. Computational simulations based on a microstructurally-motivated model of a chain of lymphangions informed from biaxial testing of isolated vessels was utilized to provide insight into the pumping mechanisms responsible for the pressure increases observed in vivo. These models suggest that the number of lympangions in the chain and smooth muscle cell force generation play a significant role in determining the maximum outflow pressure, while frequency of contraction has no effect. In vivo administration of nitric oxide (NO) attenuates lymphatic contraction, subsequently lowering the effective pumping pressure. Computational simulations suggest the reduction in contractile strength of smooth muscle cells in the presence of NO can account for the reductions in outflow pressure observed along the lymphangion chain in vivo. Thus combining computation modeling with multiple measurements of lymphatic pumping pressure provides a method for approximating intrinsic lymphatic muscle activity non-invasively in vivo, while also providing insight into factors that determine the extent that a lymphangion chain can transport fluid against an adverse pressure gradient.

Leukotriene B4 antagonism ameliorates experimental lymphedema

Science Translational Medicine

Acquired lymphedema is a cancer sequela and a global health problem currently lacking pharmacologic therapy. We have previously demonstrated that ketoprofen, an anti-inflammatory agent with dual 5-lipoxygenase and cyclooxygenase inhibitory properties, effectively reverses histopathology in experimental lymphedema. We show that the therapeutic benefit of ketoprofen is specifically attributable to its inhibition of the 5-lipoxygenase metabolite leukotriene B4 (LTB4). LTB4 antagonism reversed edema, improved lymphatic function, and restored lymphatic architecture in the murine tail model of lymphedema. In vitro, LTB4 was functionally bimodal: Lower LTB4 concentrations promoted human lymphatic endothelial cell sprouting and growth, but higher concentrations inhibited lymphangiogenesis and induced apoptosis. During lymphedema progression, lymphatic fluid LTB4 concentrations rose from initial prolymphangiogenic concentrations into an antilymphangiogenic range. LTB4 biosynthesis was similarly elevated in lymphedema patients. Low concentrations of LTB4 stimulated, whereas high concentrations of LTB4 inhibited, vascular endothelial growth factor receptor 3 and Notch pathways in cultured human lymphatic endothelial cells. Lymphatic-specific Notch1−/− mice were refractory to the beneficial effects of LTB4 antagonism, suggesting that LTB4 suppression of Notch signaling is an important mechanism in disease maintenance. In summary, we found that LTB4 was harmful to lymphatic repair at the concentrations observed in established disease. Our findings suggest that LTB4 is a promising drug target for the treatment of acquired lymphedema.

Post-prandial lymphatic pump function after a high-fat meal: a characterization of contractility, flow, and viscosity

American Journal of Physiology - Gastrointestinal and Liver Physiology

Dietary lipids are transported from the intestine through contractile lymphatics. Chronic lipid loads can adversely affect lymphatic function. However, the acute lymphatic pump response in the mesentery to a postprandial lipid meal has gone unexplored. In this study, we used the rat mesenteric collecting vessel as an in vivo model to quantify the effect of lipoproteins on vessel function. Lipid load was continuously monitored by using the intensity of a fluorescent fatty-acid analog, which we infused along with a fat emulsion through a duodenal cannula. The vessel contractility was simultaneously quantified. We demonstrated for the first time that collecting lymphatic vessels respond to an acute lipid load by reducing pump function. High lipid levels decreased contraction frequency and amplitude. We also showed a strong tonic response through a reduction in the end-diastolic and systolic diameters. We further characterized the changes in flow rate and viscosity and showed that both increase postprandially. In addition, shear-mediated Ca(2+) signaling in lymphatic endothelial cells differed when cultured with lipoproteins. Together these results show that the in vivo response could be both shear and lipid mediated and provide the first evidence that high postprandial lipid has an immediate negative effect on lymphatic function even in the acute setting.

Mechanotransduction activates Canonical Wnt/β-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves

Genes and Development

Lymphatic vasculature regulates fluid homeostasis by returning interstitial fluid to blood circulation. Lymphatic endothelial cells (LECs) are the building blocks of the entire lymphatic vasculature. LECs originate as a homogeneous population of cells predominantly from the embryonic veins and undergo stepwise morphogenesis to become the lymphatic capillaries, collecting vessels or valves. The molecular mechanisms underlying the morphogenesis of the lymphatic vasculature remain to be fully understood. Here we show that canonical Wnt/β-catenin signaling is necessary for lymphatic vascular morphogenesis. Lymphatic vascular-specific ablation of β-catenin in mice prevents the formation of lymphatic and lymphovenous valves. Additionally, lymphatic vessel patterning is defective in these mice, with abnormal recruitment of mural cells. We found that oscillatory shear stress (OSS), which promotes lymphatic vessel maturation, triggers Wnt/β-catenin signaling in LECs. In turn, Wnt/β-catenin signaling controls the expression of several molecules, including the lymphedema-associated transcription factor FOXC2. Importantly, FOXC2 completely rescues the lymphatic vessel patterning defects in mice lacking β-catenin. Thus, our work reveals that mechanical stimulation is a critical regulator of lymphatic vascular development via activation of Wnt/β-catenin signaling and, in turn, FOXC2.

The relationship between lymphangion length and maximum pressure generation established through in vivo imaging and computational modeling

American Journal of Physiology

The intrinsic contraction of collecting lymphatic vessels serves as a pumping system to propel lymph against hydrostatic pressure gradients as it returns interstitial fluid to the venous circulation. We propose and validate that the maximum opposing outflow pressure along a chain of lymphangions at which flow can be achieved increases with the length of chain. Using minimally invasive near-infrared imaging to measure the effective pumping pressure at various locations in the rat tail, we demonstrate increases in the pumping pressure along the length of the tail. Computational simulations based on a microstructurally-motivated model of a chain of lymphangions informed from biaxial testing of isolated vessels was utilized to provide insight into the pumping mechanisms responsible for the pressure increases observed in vivo. These models suggest that the number of lympangions in the chain and smooth muscle cell force generation play a significant role in determining the maximum outflow pressure, while frequency of contraction has no effect. In vivo administration of nitric oxide (NO) attenuates lymphatic contraction, subsequently lowering the effective pumping pressure. Computational simulations suggest the reduction in contractile strength of smooth muscle cells in the presence of NO can account for the reductions in outflow pressure observed along the lymphangion chain in vivo. Thus combining computation modeling with multiple measurements of lymphatic pumping pressure provides a method for approximating intrinsic lymphatic muscle activity non-invasively in vivo, while also providing insight into factors that determine the extent that a lymphangion chain can transport fluid against an adverse pressure gradient.

Leukotriene B4 antagonism ameliorates experimental lymphedema

Science Translational Medicine

Acquired lymphedema is a cancer sequela and a global health problem currently lacking pharmacologic therapy. We have previously demonstrated that ketoprofen, an anti-inflammatory agent with dual 5-lipoxygenase and cyclooxygenase inhibitory properties, effectively reverses histopathology in experimental lymphedema. We show that the therapeutic benefit of ketoprofen is specifically attributable to its inhibition of the 5-lipoxygenase metabolite leukotriene B4 (LTB4). LTB4 antagonism reversed edema, improved lymphatic function, and restored lymphatic architecture in the murine tail model of lymphedema. In vitro, LTB4 was functionally bimodal: Lower LTB4 concentrations promoted human lymphatic endothelial cell sprouting and growth, but higher concentrations inhibited lymphangiogenesis and induced apoptosis. During lymphedema progression, lymphatic fluid LTB4 concentrations rose from initial prolymphangiogenic concentrations into an antilymphangiogenic range. LTB4 biosynthesis was similarly elevated in lymphedema patients. Low concentrations of LTB4 stimulated, whereas high concentrations of LTB4 inhibited, vascular endothelial growth factor receptor 3 and Notch pathways in cultured human lymphatic endothelial cells. Lymphatic-specific Notch1−/− mice were refractory to the beneficial effects of LTB4 antagonism, suggesting that LTB4 suppression of Notch signaling is an important mechanism in disease maintenance. In summary, we found that LTB4 was harmful to lymphatic repair at the concentrations observed in established disease. Our findings suggest that LTB4 is a promising drug target for the treatment of acquired lymphedema.

Post-prandial lymphatic pump function after a high-fat meal: a characterization of contractility, flow, and viscosity

American Journal of Physiology - Gastrointestinal and Liver Physiology

Dietary lipids are transported from the intestine through contractile lymphatics. Chronic lipid loads can adversely affect lymphatic function. However, the acute lymphatic pump response in the mesentery to a postprandial lipid meal has gone unexplored. In this study, we used the rat mesenteric collecting vessel as an in vivo model to quantify the effect of lipoproteins on vessel function. Lipid load was continuously monitored by using the intensity of a fluorescent fatty-acid analog, which we infused along with a fat emulsion through a duodenal cannula. The vessel contractility was simultaneously quantified. We demonstrated for the first time that collecting lymphatic vessels respond to an acute lipid load by reducing pump function. High lipid levels decreased contraction frequency and amplitude. We also showed a strong tonic response through a reduction in the end-diastolic and systolic diameters. We further characterized the changes in flow rate and viscosity and showed that both increase postprandially. In addition, shear-mediated Ca(2+) signaling in lymphatic endothelial cells differed when cultured with lipoproteins. Together these results show that the in vivo response could be both shear and lipid mediated and provide the first evidence that high postprandial lipid has an immediate negative effect on lymphatic function even in the acute setting.

Mechanotransduction activates Canonical Wnt/β-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves

Genes and Development

Lymphatic vasculature regulates fluid homeostasis by returning interstitial fluid to blood circulation. Lymphatic endothelial cells (LECs) are the building blocks of the entire lymphatic vasculature. LECs originate as a homogeneous population of cells predominantly from the embryonic veins and undergo stepwise morphogenesis to become the lymphatic capillaries, collecting vessels or valves. The molecular mechanisms underlying the morphogenesis of the lymphatic vasculature remain to be fully understood. Here we show that canonical Wnt/β-catenin signaling is necessary for lymphatic vascular morphogenesis. Lymphatic vascular-specific ablation of β-catenin in mice prevents the formation of lymphatic and lymphovenous valves. Additionally, lymphatic vessel patterning is defective in these mice, with abnormal recruitment of mural cells. We found that oscillatory shear stress (OSS), which promotes lymphatic vessel maturation, triggers Wnt/β-catenin signaling in LECs. In turn, Wnt/β-catenin signaling controls the expression of several molecules, including the lymphedema-associated transcription factor FOXC2. Importantly, FOXC2 completely rescues the lymphatic vessel patterning defects in mice lacking β-catenin. Thus, our work reveals that mechanical stimulation is a critical regulator of lymphatic vascular development via activation of Wnt/β-catenin signaling and, in turn, FOXC2.

Use of a novel portable three-dimensional scanner to measure limb volume and circumference in patients with filarial lymphedema

American Journal of Tropical Medicine and Hygeine

The World Health Organization’s Global Program to Eliminate Lymphatic Filariasis (LF) has reduced LF transmission worldwide, but millions remain affected by filarial lymphedema. Tools for clinically monitoring lymphedema in developing nations are limited. We tested a novel, portable, infrared three-dimensional (3D) scanner against water displacement (WD) and tape measurement of limb circumference (TMLC) among patients with filarial leg lymphedema in Galle, Sri Lanka. Outcomes were accuracy and reproducibility of scanner measurements. In parallel, we also tested the reproducibility of skin thickness ultrasound (STU) measurements. We examined 52 patients (104 limbs) with lymphedema of stages 0–6 (N = 28, 19, 20, 21, 2, 4, and 10, respectively). Scanner measurements correlated nearly perfectly with WD (r 2 = 0.9945) and TMLC values (r 2 > 0.9801). The average time required to acquire scanner measurements for both legs was 2.2 minutes, compared with 17.4, 7.5, and 31.7 minutes, respectively, for WD, TMLC, and STU. Median interexaminer coefficients of variation (CVs) for volume measurements were 1.1% (interquartile range [IQR] 0.5–2.1%) for WD and 1.7% (IQR 1.2–2.4%) for the 3D scanner. CVs for circumference measurements were 1.4% (IQR 0.8–2.4%) by TMLC and 1.3% (0.8–1.9%) by the 3D scanner. Median interexaminer CV for STU was 13.7% (IQR 8.5–21.3%). The portable 3D scanner noninvasively provided accurate and reproducible limb volume and circumference measurements in approximately 2 minutes per patient. This portable technology has the potential to greatly improve assessment and monitoring of lymphedema in the clinic and in the field.

The relationship between lymphangion length and maximum pressure generation established through in vivo imaging and computational modeling

American Journal of Physiology

The intrinsic contraction of collecting lymphatic vessels serves as a pumping system to propel lymph against hydrostatic pressure gradients as it returns interstitial fluid to the venous circulation. We propose and validate that the maximum opposing outflow pressure along a chain of lymphangions at which flow can be achieved increases with the length of chain. Using minimally invasive near-infrared imaging to measure the effective pumping pressure at various locations in the rat tail, we demonstrate increases in the pumping pressure along the length of the tail. Computational simulations based on a microstructurally-motivated model of a chain of lymphangions informed from biaxial testing of isolated vessels was utilized to provide insight into the pumping mechanisms responsible for the pressure increases observed in vivo. These models suggest that the number of lympangions in the chain and smooth muscle cell force generation play a significant role in determining the maximum outflow pressure, while frequency of contraction has no effect. In vivo administration of nitric oxide (NO) attenuates lymphatic contraction, subsequently lowering the effective pumping pressure. Computational simulations suggest the reduction in contractile strength of smooth muscle cells in the presence of NO can account for the reductions in outflow pressure observed along the lymphangion chain in vivo. Thus combining computation modeling with multiple measurements of lymphatic pumping pressure provides a method for approximating intrinsic lymphatic muscle activity non-invasively in vivo, while also providing insight into factors that determine the extent that a lymphangion chain can transport fluid against an adverse pressure gradient.

Leukotriene B4 antagonism ameliorates experimental lymphedema

Science Translational Medicine

Acquired lymphedema is a cancer sequela and a global health problem currently lacking pharmacologic therapy. We have previously demonstrated that ketoprofen, an anti-inflammatory agent with dual 5-lipoxygenase and cyclooxygenase inhibitory properties, effectively reverses histopathology in experimental lymphedema. We show that the therapeutic benefit of ketoprofen is specifically attributable to its inhibition of the 5-lipoxygenase metabolite leukotriene B4 (LTB4). LTB4 antagonism reversed edema, improved lymphatic function, and restored lymphatic architecture in the murine tail model of lymphedema. In vitro, LTB4 was functionally bimodal: Lower LTB4 concentrations promoted human lymphatic endothelial cell sprouting and growth, but higher concentrations inhibited lymphangiogenesis and induced apoptosis. During lymphedema progression, lymphatic fluid LTB4 concentrations rose from initial prolymphangiogenic concentrations into an antilymphangiogenic range. LTB4 biosynthesis was similarly elevated in lymphedema patients. Low concentrations of LTB4 stimulated, whereas high concentrations of LTB4 inhibited, vascular endothelial growth factor receptor 3 and Notch pathways in cultured human lymphatic endothelial cells. Lymphatic-specific Notch1−/− mice were refractory to the beneficial effects of LTB4 antagonism, suggesting that LTB4 suppression of Notch signaling is an important mechanism in disease maintenance. In summary, we found that LTB4 was harmful to lymphatic repair at the concentrations observed in established disease. Our findings suggest that LTB4 is a promising drug target for the treatment of acquired lymphedema.

Post-prandial lymphatic pump function after a high-fat meal: a characterization of contractility, flow, and viscosity

American Journal of Physiology - Gastrointestinal and Liver Physiology

Dietary lipids are transported from the intestine through contractile lymphatics. Chronic lipid loads can adversely affect lymphatic function. However, the acute lymphatic pump response in the mesentery to a postprandial lipid meal has gone unexplored. In this study, we used the rat mesenteric collecting vessel as an in vivo model to quantify the effect of lipoproteins on vessel function. Lipid load was continuously monitored by using the intensity of a fluorescent fatty-acid analog, which we infused along with a fat emulsion through a duodenal cannula. The vessel contractility was simultaneously quantified. We demonstrated for the first time that collecting lymphatic vessels respond to an acute lipid load by reducing pump function. High lipid levels decreased contraction frequency and amplitude. We also showed a strong tonic response through a reduction in the end-diastolic and systolic diameters. We further characterized the changes in flow rate and viscosity and showed that both increase postprandially. In addition, shear-mediated Ca(2+) signaling in lymphatic endothelial cells differed when cultured with lipoproteins. Together these results show that the in vivo response could be both shear and lipid mediated and provide the first evidence that high postprandial lipid has an immediate negative effect on lymphatic function even in the acute setting.

Mechanotransduction activates Canonical Wnt/β-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves

Genes and Development

Lymphatic vasculature regulates fluid homeostasis by returning interstitial fluid to blood circulation. Lymphatic endothelial cells (LECs) are the building blocks of the entire lymphatic vasculature. LECs originate as a homogeneous population of cells predominantly from the embryonic veins and undergo stepwise morphogenesis to become the lymphatic capillaries, collecting vessels or valves. The molecular mechanisms underlying the morphogenesis of the lymphatic vasculature remain to be fully understood. Here we show that canonical Wnt/β-catenin signaling is necessary for lymphatic vascular morphogenesis. Lymphatic vascular-specific ablation of β-catenin in mice prevents the formation of lymphatic and lymphovenous valves. Additionally, lymphatic vessel patterning is defective in these mice, with abnormal recruitment of mural cells. We found that oscillatory shear stress (OSS), which promotes lymphatic vessel maturation, triggers Wnt/β-catenin signaling in LECs. In turn, Wnt/β-catenin signaling controls the expression of several molecules, including the lymphedema-associated transcription factor FOXC2. Importantly, FOXC2 completely rescues the lymphatic vessel patterning defects in mice lacking β-catenin. Thus, our work reveals that mechanical stimulation is a critical regulator of lymphatic vascular development via activation of Wnt/β-catenin signaling and, in turn, FOXC2.

Use of a novel portable three-dimensional scanner to measure limb volume and circumference in patients with filarial lymphedema

American Journal of Tropical Medicine and Hygeine

The World Health Organization’s Global Program to Eliminate Lymphatic Filariasis (LF) has reduced LF transmission worldwide, but millions remain affected by filarial lymphedema. Tools for clinically monitoring lymphedema in developing nations are limited. We tested a novel, portable, infrared three-dimensional (3D) scanner against water displacement (WD) and tape measurement of limb circumference (TMLC) among patients with filarial leg lymphedema in Galle, Sri Lanka. Outcomes were accuracy and reproducibility of scanner measurements. In parallel, we also tested the reproducibility of skin thickness ultrasound (STU) measurements. We examined 52 patients (104 limbs) with lymphedema of stages 0–6 (N = 28, 19, 20, 21, 2, 4, and 10, respectively). Scanner measurements correlated nearly perfectly with WD (r 2 = 0.9945) and TMLC values (r 2 > 0.9801). The average time required to acquire scanner measurements for both legs was 2.2 minutes, compared with 17.4, 7.5, and 31.7 minutes, respectively, for WD, TMLC, and STU. Median interexaminer coefficients of variation (CVs) for volume measurements were 1.1% (interquartile range [IQR] 0.5–2.1%) for WD and 1.7% (IQR 1.2–2.4%) for the 3D scanner. CVs for circumference measurements were 1.4% (IQR 0.8–2.4%) by TMLC and 1.3% (0.8–1.9%) by the 3D scanner. Median interexaminer CV for STU was 13.7% (IQR 8.5–21.3%). The portable 3D scanner noninvasively provided accurate and reproducible limb volume and circumference measurements in approximately 2 minutes per patient. This portable technology has the potential to greatly improve assessment and monitoring of lymphedema in the clinic and in the field.

Optimization of culture and analysis methods for enhancing long-term B. malayi survival, molting, and motility in vitro

Open Parasitology

Lymphatic filariasis is a neglected tropical disease caused by roundworm parasites such as Brugia malayi that spread via a mosquito vector. In vitro culture of these parasites provides controlled conditions to understand parasite biology and provides a cheaper way to screen potential micro- and macrofilaricides. Published studies have used a wide array of approaches and metrics regarding in vitro cultures of Brugia malayi; as a result, drawing comparisons and identifying the reasons why inability to reproduce outcomes are difficult. This study sought to determine conditions that ensure reproducible outcomes and used evaluation metrics that are easily measured and can be automated to ensure objectivity. We found culturing Brugia malayi L3 in endothelial basal media supplemented with 20% fetal bovine serum and 75 µM ascorbic acid in a temperature and humidity controlled incubator produced better survival and molting rates as well as longer and more motile parasites than previously reported. The benefit of ascorbic acid seemed to be unique to L3 parasites, as the addition of ascorbic acid to adult parasites had no significant impact on survival or motility. The methods reported in this study will help in designing experiments for both parasite behavior studies and drug screening applications for disease eradication.

The relationship between lymphangion length and maximum pressure generation established through in vivo imaging and computational modeling

American Journal of Physiology

The intrinsic contraction of collecting lymphatic vessels serves as a pumping system to propel lymph against hydrostatic pressure gradients as it returns interstitial fluid to the venous circulation. We propose and validate that the maximum opposing outflow pressure along a chain of lymphangions at which flow can be achieved increases with the length of chain. Using minimally invasive near-infrared imaging to measure the effective pumping pressure at various locations in the rat tail, we demonstrate increases in the pumping pressure along the length of the tail. Computational simulations based on a microstructurally-motivated model of a chain of lymphangions informed from biaxial testing of isolated vessels was utilized to provide insight into the pumping mechanisms responsible for the pressure increases observed in vivo. These models suggest that the number of lympangions in the chain and smooth muscle cell force generation play a significant role in determining the maximum outflow pressure, while frequency of contraction has no effect. In vivo administration of nitric oxide (NO) attenuates lymphatic contraction, subsequently lowering the effective pumping pressure. Computational simulations suggest the reduction in contractile strength of smooth muscle cells in the presence of NO can account for the reductions in outflow pressure observed along the lymphangion chain in vivo. Thus combining computation modeling with multiple measurements of lymphatic pumping pressure provides a method for approximating intrinsic lymphatic muscle activity non-invasively in vivo, while also providing insight into factors that determine the extent that a lymphangion chain can transport fluid against an adverse pressure gradient.

Leukotriene B4 antagonism ameliorates experimental lymphedema

Science Translational Medicine

Acquired lymphedema is a cancer sequela and a global health problem currently lacking pharmacologic therapy. We have previously demonstrated that ketoprofen, an anti-inflammatory agent with dual 5-lipoxygenase and cyclooxygenase inhibitory properties, effectively reverses histopathology in experimental lymphedema. We show that the therapeutic benefit of ketoprofen is specifically attributable to its inhibition of the 5-lipoxygenase metabolite leukotriene B4 (LTB4). LTB4 antagonism reversed edema, improved lymphatic function, and restored lymphatic architecture in the murine tail model of lymphedema. In vitro, LTB4 was functionally bimodal: Lower LTB4 concentrations promoted human lymphatic endothelial cell sprouting and growth, but higher concentrations inhibited lymphangiogenesis and induced apoptosis. During lymphedema progression, lymphatic fluid LTB4 concentrations rose from initial prolymphangiogenic concentrations into an antilymphangiogenic range. LTB4 biosynthesis was similarly elevated in lymphedema patients. Low concentrations of LTB4 stimulated, whereas high concentrations of LTB4 inhibited, vascular endothelial growth factor receptor 3 and Notch pathways in cultured human lymphatic endothelial cells. Lymphatic-specific Notch1−/− mice were refractory to the beneficial effects of LTB4 antagonism, suggesting that LTB4 suppression of Notch signaling is an important mechanism in disease maintenance. In summary, we found that LTB4 was harmful to lymphatic repair at the concentrations observed in established disease. Our findings suggest that LTB4 is a promising drug target for the treatment of acquired lymphedema.

Post-prandial lymphatic pump function after a high-fat meal: a characterization of contractility, flow, and viscosity

American Journal of Physiology - Gastrointestinal and Liver Physiology

Dietary lipids are transported from the intestine through contractile lymphatics. Chronic lipid loads can adversely affect lymphatic function. However, the acute lymphatic pump response in the mesentery to a postprandial lipid meal has gone unexplored. In this study, we used the rat mesenteric collecting vessel as an in vivo model to quantify the effect of lipoproteins on vessel function. Lipid load was continuously monitored by using the intensity of a fluorescent fatty-acid analog, which we infused along with a fat emulsion through a duodenal cannula. The vessel contractility was simultaneously quantified. We demonstrated for the first time that collecting lymphatic vessels respond to an acute lipid load by reducing pump function. High lipid levels decreased contraction frequency and amplitude. We also showed a strong tonic response through a reduction in the end-diastolic and systolic diameters. We further characterized the changes in flow rate and viscosity and showed that both increase postprandially. In addition, shear-mediated Ca(2+) signaling in lymphatic endothelial cells differed when cultured with lipoproteins. Together these results show that the in vivo response could be both shear and lipid mediated and provide the first evidence that high postprandial lipid has an immediate negative effect on lymphatic function even in the acute setting.

Mechanotransduction activates Canonical Wnt/β-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves

Genes and Development

Lymphatic vasculature regulates fluid homeostasis by returning interstitial fluid to blood circulation. Lymphatic endothelial cells (LECs) are the building blocks of the entire lymphatic vasculature. LECs originate as a homogeneous population of cells predominantly from the embryonic veins and undergo stepwise morphogenesis to become the lymphatic capillaries, collecting vessels or valves. The molecular mechanisms underlying the morphogenesis of the lymphatic vasculature remain to be fully understood. Here we show that canonical Wnt/β-catenin signaling is necessary for lymphatic vascular morphogenesis. Lymphatic vascular-specific ablation of β-catenin in mice prevents the formation of lymphatic and lymphovenous valves. Additionally, lymphatic vessel patterning is defective in these mice, with abnormal recruitment of mural cells. We found that oscillatory shear stress (OSS), which promotes lymphatic vessel maturation, triggers Wnt/β-catenin signaling in LECs. In turn, Wnt/β-catenin signaling controls the expression of several molecules, including the lymphedema-associated transcription factor FOXC2. Importantly, FOXC2 completely rescues the lymphatic vessel patterning defects in mice lacking β-catenin. Thus, our work reveals that mechanical stimulation is a critical regulator of lymphatic vascular development via activation of Wnt/β-catenin signaling and, in turn, FOXC2.

Use of a novel portable three-dimensional scanner to measure limb volume and circumference in patients with filarial lymphedema

American Journal of Tropical Medicine and Hygeine

The World Health Organization’s Global Program to Eliminate Lymphatic Filariasis (LF) has reduced LF transmission worldwide, but millions remain affected by filarial lymphedema. Tools for clinically monitoring lymphedema in developing nations are limited. We tested a novel, portable, infrared three-dimensional (3D) scanner against water displacement (WD) and tape measurement of limb circumference (TMLC) among patients with filarial leg lymphedema in Galle, Sri Lanka. Outcomes were accuracy and reproducibility of scanner measurements. In parallel, we also tested the reproducibility of skin thickness ultrasound (STU) measurements. We examined 52 patients (104 limbs) with lymphedema of stages 0–6 (N = 28, 19, 20, 21, 2, 4, and 10, respectively). Scanner measurements correlated nearly perfectly with WD (r 2 = 0.9945) and TMLC values (r 2 > 0.9801). The average time required to acquire scanner measurements for both legs was 2.2 minutes, compared with 17.4, 7.5, and 31.7 minutes, respectively, for WD, TMLC, and STU. Median interexaminer coefficients of variation (CVs) for volume measurements were 1.1% (interquartile range [IQR] 0.5–2.1%) for WD and 1.7% (IQR 1.2–2.4%) for the 3D scanner. CVs for circumference measurements were 1.4% (IQR 0.8–2.4%) by TMLC and 1.3% (0.8–1.9%) by the 3D scanner. Median interexaminer CV for STU was 13.7% (IQR 8.5–21.3%). The portable 3D scanner noninvasively provided accurate and reproducible limb volume and circumference measurements in approximately 2 minutes per patient. This portable technology has the potential to greatly improve assessment and monitoring of lymphedema in the clinic and in the field.

Optimization of culture and analysis methods for enhancing long-term B. malayi survival, molting, and motility in vitro

Open Parasitology

Lymphatic filariasis is a neglected tropical disease caused by roundworm parasites such as Brugia malayi that spread via a mosquito vector. In vitro culture of these parasites provides controlled conditions to understand parasite biology and provides a cheaper way to screen potential micro- and macrofilaricides. Published studies have used a wide array of approaches and metrics regarding in vitro cultures of Brugia malayi; as a result, drawing comparisons and identifying the reasons why inability to reproduce outcomes are difficult. This study sought to determine conditions that ensure reproducible outcomes and used evaluation metrics that are easily measured and can be automated to ensure objectivity. We found culturing Brugia malayi L3 in endothelial basal media supplemented with 20% fetal bovine serum and 75 µM ascorbic acid in a temperature and humidity controlled incubator produced better survival and molting rates as well as longer and more motile parasites than previously reported. The benefit of ascorbic acid seemed to be unique to L3 parasites, as the addition of ascorbic acid to adult parasites had no significant impact on survival or motility. The methods reported in this study will help in designing experiments for both parasite behavior studies and drug screening applications for disease eradication.

Experimental models used to assess lymphatic contractile function

Lymphatic Research and Biology

The relationship between lymphangion length and maximum pressure generation established through in vivo imaging and computational modeling

American Journal of Physiology

The intrinsic contraction of collecting lymphatic vessels serves as a pumping system to propel lymph against hydrostatic pressure gradients as it returns interstitial fluid to the venous circulation. We propose and validate that the maximum opposing outflow pressure along a chain of lymphangions at which flow can be achieved increases with the length of chain. Using minimally invasive near-infrared imaging to measure the effective pumping pressure at various locations in the rat tail, we demonstrate increases in the pumping pressure along the length of the tail. Computational simulations based on a microstructurally-motivated model of a chain of lymphangions informed from biaxial testing of isolated vessels was utilized to provide insight into the pumping mechanisms responsible for the pressure increases observed in vivo. These models suggest that the number of lympangions in the chain and smooth muscle cell force generation play a significant role in determining the maximum outflow pressure, while frequency of contraction has no effect. In vivo administration of nitric oxide (NO) attenuates lymphatic contraction, subsequently lowering the effective pumping pressure. Computational simulations suggest the reduction in contractile strength of smooth muscle cells in the presence of NO can account for the reductions in outflow pressure observed along the lymphangion chain in vivo. Thus combining computation modeling with multiple measurements of lymphatic pumping pressure provides a method for approximating intrinsic lymphatic muscle activity non-invasively in vivo, while also providing insight into factors that determine the extent that a lymphangion chain can transport fluid against an adverse pressure gradient.

Leukotriene B4 antagonism ameliorates experimental lymphedema

Science Translational Medicine

Acquired lymphedema is a cancer sequela and a global health problem currently lacking pharmacologic therapy. We have previously demonstrated that ketoprofen, an anti-inflammatory agent with dual 5-lipoxygenase and cyclooxygenase inhibitory properties, effectively reverses histopathology in experimental lymphedema. We show that the therapeutic benefit of ketoprofen is specifically attributable to its inhibition of the 5-lipoxygenase metabolite leukotriene B4 (LTB4). LTB4 antagonism reversed edema, improved lymphatic function, and restored lymphatic architecture in the murine tail model of lymphedema. In vitro, LTB4 was functionally bimodal: Lower LTB4 concentrations promoted human lymphatic endothelial cell sprouting and growth, but higher concentrations inhibited lymphangiogenesis and induced apoptosis. During lymphedema progression, lymphatic fluid LTB4 concentrations rose from initial prolymphangiogenic concentrations into an antilymphangiogenic range. LTB4 biosynthesis was similarly elevated in lymphedema patients. Low concentrations of LTB4 stimulated, whereas high concentrations of LTB4 inhibited, vascular endothelial growth factor receptor 3 and Notch pathways in cultured human lymphatic endothelial cells. Lymphatic-specific Notch1−/− mice were refractory to the beneficial effects of LTB4 antagonism, suggesting that LTB4 suppression of Notch signaling is an important mechanism in disease maintenance. In summary, we found that LTB4 was harmful to lymphatic repair at the concentrations observed in established disease. Our findings suggest that LTB4 is a promising drug target for the treatment of acquired lymphedema.

Post-prandial lymphatic pump function after a high-fat meal: a characterization of contractility, flow, and viscosity

American Journal of Physiology - Gastrointestinal and Liver Physiology

Dietary lipids are transported from the intestine through contractile lymphatics. Chronic lipid loads can adversely affect lymphatic function. However, the acute lymphatic pump response in the mesentery to a postprandial lipid meal has gone unexplored. In this study, we used the rat mesenteric collecting vessel as an in vivo model to quantify the effect of lipoproteins on vessel function. Lipid load was continuously monitored by using the intensity of a fluorescent fatty-acid analog, which we infused along with a fat emulsion through a duodenal cannula. The vessel contractility was simultaneously quantified. We demonstrated for the first time that collecting lymphatic vessels respond to an acute lipid load by reducing pump function. High lipid levels decreased contraction frequency and amplitude. We also showed a strong tonic response through a reduction in the end-diastolic and systolic diameters. We further characterized the changes in flow rate and viscosity and showed that both increase postprandially. In addition, shear-mediated Ca(2+) signaling in lymphatic endothelial cells differed when cultured with lipoproteins. Together these results show that the in vivo response could be both shear and lipid mediated and provide the first evidence that high postprandial lipid has an immediate negative effect on lymphatic function even in the acute setting.

Mechanotransduction activates Canonical Wnt/β-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves

Genes and Development

Lymphatic vasculature regulates fluid homeostasis by returning interstitial fluid to blood circulation. Lymphatic endothelial cells (LECs) are the building blocks of the entire lymphatic vasculature. LECs originate as a homogeneous population of cells predominantly from the embryonic veins and undergo stepwise morphogenesis to become the lymphatic capillaries, collecting vessels or valves. The molecular mechanisms underlying the morphogenesis of the lymphatic vasculature remain to be fully understood. Here we show that canonical Wnt/β-catenin signaling is necessary for lymphatic vascular morphogenesis. Lymphatic vascular-specific ablation of β-catenin in mice prevents the formation of lymphatic and lymphovenous valves. Additionally, lymphatic vessel patterning is defective in these mice, with abnormal recruitment of mural cells. We found that oscillatory shear stress (OSS), which promotes lymphatic vessel maturation, triggers Wnt/β-catenin signaling in LECs. In turn, Wnt/β-catenin signaling controls the expression of several molecules, including the lymphedema-associated transcription factor FOXC2. Importantly, FOXC2 completely rescues the lymphatic vessel patterning defects in mice lacking β-catenin. Thus, our work reveals that mechanical stimulation is a critical regulator of lymphatic vascular development via activation of Wnt/β-catenin signaling and, in turn, FOXC2.

Use of a novel portable three-dimensional scanner to measure limb volume and circumference in patients with filarial lymphedema

American Journal of Tropical Medicine and Hygeine

The World Health Organization’s Global Program to Eliminate Lymphatic Filariasis (LF) has reduced LF transmission worldwide, but millions remain affected by filarial lymphedema. Tools for clinically monitoring lymphedema in developing nations are limited. We tested a novel, portable, infrared three-dimensional (3D) scanner against water displacement (WD) and tape measurement of limb circumference (TMLC) among patients with filarial leg lymphedema in Galle, Sri Lanka. Outcomes were accuracy and reproducibility of scanner measurements. In parallel, we also tested the reproducibility of skin thickness ultrasound (STU) measurements. We examined 52 patients (104 limbs) with lymphedema of stages 0–6 (N = 28, 19, 20, 21, 2, 4, and 10, respectively). Scanner measurements correlated nearly perfectly with WD (r 2 = 0.9945) and TMLC values (r 2 > 0.9801). The average time required to acquire scanner measurements for both legs was 2.2 minutes, compared with 17.4, 7.5, and 31.7 minutes, respectively, for WD, TMLC, and STU. Median interexaminer coefficients of variation (CVs) for volume measurements were 1.1% (interquartile range [IQR] 0.5–2.1%) for WD and 1.7% (IQR 1.2–2.4%) for the 3D scanner. CVs for circumference measurements were 1.4% (IQR 0.8–2.4%) by TMLC and 1.3% (0.8–1.9%) by the 3D scanner. Median interexaminer CV for STU was 13.7% (IQR 8.5–21.3%). The portable 3D scanner noninvasively provided accurate and reproducible limb volume and circumference measurements in approximately 2 minutes per patient. This portable technology has the potential to greatly improve assessment and monitoring of lymphedema in the clinic and in the field.

Optimization of culture and analysis methods for enhancing long-term B. malayi survival, molting, and motility in vitro

Open Parasitology

Lymphatic filariasis is a neglected tropical disease caused by roundworm parasites such as Brugia malayi that spread via a mosquito vector. In vitro culture of these parasites provides controlled conditions to understand parasite biology and provides a cheaper way to screen potential micro- and macrofilaricides. Published studies have used a wide array of approaches and metrics regarding in vitro cultures of Brugia malayi; as a result, drawing comparisons and identifying the reasons why inability to reproduce outcomes are difficult. This study sought to determine conditions that ensure reproducible outcomes and used evaluation metrics that are easily measured and can be automated to ensure objectivity. We found culturing Brugia malayi L3 in endothelial basal media supplemented with 20% fetal bovine serum and 75 µM ascorbic acid in a temperature and humidity controlled incubator produced better survival and molting rates as well as longer and more motile parasites than previously reported. The benefit of ascorbic acid seemed to be unique to L3 parasites, as the addition of ascorbic acid to adult parasites had no significant impact on survival or motility. The methods reported in this study will help in designing experiments for both parasite behavior studies and drug screening applications for disease eradication.

Experimental models used to assess lymphatic contractile function

Lymphatic Research and Biology

TLR-exosomes exhibit distinct kinetics and effector function

Scientific Reports

The innate immune system is vital to rapidly responding to pathogens and Toll-like receptors (TLRs) are a critical component of this response. Nanovesicular exosomes play a role in immunity, but to date their exact contribution to the dissemination of the TLR response is unknown. Here we show that exosomes from TLR stimulated cells can largely recapitulate TLR activation in distal cells in vitro. We can abrogate the action-at-a-distance signaling of exosomes by UV irradiation, demonstrating that RNA is crucial for their effector function. We are the first to show that exosomes derived from poly(I:C) stimulated cells induce in vivo macrophage M1-like polarization within murine lymph nodes. These poly(I:C) exosomes demonstrate enhanced trafficking to the node and preferentially recruit neutrophils as compared to control exosomes. This work definitively establishes the differential effector function for exosomes in communicating the TLR activation state of the cell of origin.

The relationship between lymphangion length and maximum pressure generation established through in vivo imaging and computational modeling

American Journal of Physiology

The intrinsic contraction of collecting lymphatic vessels serves as a pumping system to propel lymph against hydrostatic pressure gradients as it returns interstitial fluid to the venous circulation. We propose and validate that the maximum opposing outflow pressure along a chain of lymphangions at which flow can be achieved increases with the length of chain. Using minimally invasive near-infrared imaging to measure the effective pumping pressure at various locations in the rat tail, we demonstrate increases in the pumping pressure along the length of the tail. Computational simulations based on a microstructurally-motivated model of a chain of lymphangions informed from biaxial testing of isolated vessels was utilized to provide insight into the pumping mechanisms responsible for the pressure increases observed in vivo. These models suggest that the number of lympangions in the chain and smooth muscle cell force generation play a significant role in determining the maximum outflow pressure, while frequency of contraction has no effect. In vivo administration of nitric oxide (NO) attenuates lymphatic contraction, subsequently lowering the effective pumping pressure. Computational simulations suggest the reduction in contractile strength of smooth muscle cells in the presence of NO can account for the reductions in outflow pressure observed along the lymphangion chain in vivo. Thus combining computation modeling with multiple measurements of lymphatic pumping pressure provides a method for approximating intrinsic lymphatic muscle activity non-invasively in vivo, while also providing insight into factors that determine the extent that a lymphangion chain can transport fluid against an adverse pressure gradient.

Leukotriene B4 antagonism ameliorates experimental lymphedema

Science Translational Medicine

Acquired lymphedema is a cancer sequela and a global health problem currently lacking pharmacologic therapy. We have previously demonstrated that ketoprofen, an anti-inflammatory agent with dual 5-lipoxygenase and cyclooxygenase inhibitory properties, effectively reverses histopathology in experimental lymphedema. We show that the therapeutic benefit of ketoprofen is specifically attributable to its inhibition of the 5-lipoxygenase metabolite leukotriene B4 (LTB4). LTB4 antagonism reversed edema, improved lymphatic function, and restored lymphatic architecture in the murine tail model of lymphedema. In vitro, LTB4 was functionally bimodal: Lower LTB4 concentrations promoted human lymphatic endothelial cell sprouting and growth, but higher concentrations inhibited lymphangiogenesis and induced apoptosis. During lymphedema progression, lymphatic fluid LTB4 concentrations rose from initial prolymphangiogenic concentrations into an antilymphangiogenic range. LTB4 biosynthesis was similarly elevated in lymphedema patients. Low concentrations of LTB4 stimulated, whereas high concentrations of LTB4 inhibited, vascular endothelial growth factor receptor 3 and Notch pathways in cultured human lymphatic endothelial cells. Lymphatic-specific Notch1−/− mice were refractory to the beneficial effects of LTB4 antagonism, suggesting that LTB4 suppression of Notch signaling is an important mechanism in disease maintenance. In summary, we found that LTB4 was harmful to lymphatic repair at the concentrations observed in established disease. Our findings suggest that LTB4 is a promising drug target for the treatment of acquired lymphedema.

Post-prandial lymphatic pump function after a high-fat meal: a characterization of contractility, flow, and viscosity

American Journal of Physiology - Gastrointestinal and Liver Physiology

Dietary lipids are transported from the intestine through contractile lymphatics. Chronic lipid loads can adversely affect lymphatic function. However, the acute lymphatic pump response in the mesentery to a postprandial lipid meal has gone unexplored. In this study, we used the rat mesenteric collecting vessel as an in vivo model to quantify the effect of lipoproteins on vessel function. Lipid load was continuously monitored by using the intensity of a fluorescent fatty-acid analog, which we infused along with a fat emulsion through a duodenal cannula. The vessel contractility was simultaneously quantified. We demonstrated for the first time that collecting lymphatic vessels respond to an acute lipid load by reducing pump function. High lipid levels decreased contraction frequency and amplitude. We also showed a strong tonic response through a reduction in the end-diastolic and systolic diameters. We further characterized the changes in flow rate and viscosity and showed that both increase postprandially. In addition, shear-mediated Ca(2+) signaling in lymphatic endothelial cells differed when cultured with lipoproteins. Together these results show that the in vivo response could be both shear and lipid mediated and provide the first evidence that high postprandial lipid has an immediate negative effect on lymphatic function even in the acute setting.

Mechanotransduction activates Canonical Wnt/β-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves

Genes and Development

Lymphatic vasculature regulates fluid homeostasis by returning interstitial fluid to blood circulation. Lymphatic endothelial cells (LECs) are the building blocks of the entire lymphatic vasculature. LECs originate as a homogeneous population of cells predominantly from the embryonic veins and undergo stepwise morphogenesis to become the lymphatic capillaries, collecting vessels or valves. The molecular mechanisms underlying the morphogenesis of the lymphatic vasculature remain to be fully understood. Here we show that canonical Wnt/β-catenin signaling is necessary for lymphatic vascular morphogenesis. Lymphatic vascular-specific ablation of β-catenin in mice prevents the formation of lymphatic and lymphovenous valves. Additionally, lymphatic vessel patterning is defective in these mice, with abnormal recruitment of mural cells. We found that oscillatory shear stress (OSS), which promotes lymphatic vessel maturation, triggers Wnt/β-catenin signaling in LECs. In turn, Wnt/β-catenin signaling controls the expression of several molecules, including the lymphedema-associated transcription factor FOXC2. Importantly, FOXC2 completely rescues the lymphatic vessel patterning defects in mice lacking β-catenin. Thus, our work reveals that mechanical stimulation is a critical regulator of lymphatic vascular development via activation of Wnt/β-catenin signaling and, in turn, FOXC2.

Use of a novel portable three-dimensional scanner to measure limb volume and circumference in patients with filarial lymphedema

American Journal of Tropical Medicine and Hygeine

The World Health Organization’s Global Program to Eliminate Lymphatic Filariasis (LF) has reduced LF transmission worldwide, but millions remain affected by filarial lymphedema. Tools for clinically monitoring lymphedema in developing nations are limited. We tested a novel, portable, infrared three-dimensional (3D) scanner against water displacement (WD) and tape measurement of limb circumference (TMLC) among patients with filarial leg lymphedema in Galle, Sri Lanka. Outcomes were accuracy and reproducibility of scanner measurements. In parallel, we also tested the reproducibility of skin thickness ultrasound (STU) measurements. We examined 52 patients (104 limbs) with lymphedema of stages 0–6 (N = 28, 19, 20, 21, 2, 4, and 10, respectively). Scanner measurements correlated nearly perfectly with WD (r 2 = 0.9945) and TMLC values (r 2 > 0.9801). The average time required to acquire scanner measurements for both legs was 2.2 minutes, compared with 17.4, 7.5, and 31.7 minutes, respectively, for WD, TMLC, and STU. Median interexaminer coefficients of variation (CVs) for volume measurements were 1.1% (interquartile range [IQR] 0.5–2.1%) for WD and 1.7% (IQR 1.2–2.4%) for the 3D scanner. CVs for circumference measurements were 1.4% (IQR 0.8–2.4%) by TMLC and 1.3% (0.8–1.9%) by the 3D scanner. Median interexaminer CV for STU was 13.7% (IQR 8.5–21.3%). The portable 3D scanner noninvasively provided accurate and reproducible limb volume and circumference measurements in approximately 2 minutes per patient. This portable technology has the potential to greatly improve assessment and monitoring of lymphedema in the clinic and in the field.

Optimization of culture and analysis methods for enhancing long-term B. malayi survival, molting, and motility in vitro

Open Parasitology

Lymphatic filariasis is a neglected tropical disease caused by roundworm parasites such as Brugia malayi that spread via a mosquito vector. In vitro culture of these parasites provides controlled conditions to understand parasite biology and provides a cheaper way to screen potential micro- and macrofilaricides. Published studies have used a wide array of approaches and metrics regarding in vitro cultures of Brugia malayi; as a result, drawing comparisons and identifying the reasons why inability to reproduce outcomes are difficult. This study sought to determine conditions that ensure reproducible outcomes and used evaluation metrics that are easily measured and can be automated to ensure objectivity. We found culturing Brugia malayi L3 in endothelial basal media supplemented with 20% fetal bovine serum and 75 µM ascorbic acid in a temperature and humidity controlled incubator produced better survival and molting rates as well as longer and more motile parasites than previously reported. The benefit of ascorbic acid seemed to be unique to L3 parasites, as the addition of ascorbic acid to adult parasites had no significant impact on survival or motility. The methods reported in this study will help in designing experiments for both parasite behavior studies and drug screening applications for disease eradication.

Experimental models used to assess lymphatic contractile function

Lymphatic Research and Biology

TLR-exosomes exhibit distinct kinetics and effector function

Scientific Reports

The innate immune system is vital to rapidly responding to pathogens and Toll-like receptors (TLRs) are a critical component of this response. Nanovesicular exosomes play a role in immunity, but to date their exact contribution to the dissemination of the TLR response is unknown. Here we show that exosomes from TLR stimulated cells can largely recapitulate TLR activation in distal cells in vitro. We can abrogate the action-at-a-distance signaling of exosomes by UV irradiation, demonstrating that RNA is crucial for their effector function. We are the first to show that exosomes derived from poly(I:C) stimulated cells induce in vivo macrophage M1-like polarization within murine lymph nodes. These poly(I:C) exosomes demonstrate enhanced trafficking to the node and preferentially recruit neutrophils as compared to control exosomes. This work definitively establishes the differential effector function for exosomes in communicating the TLR activation state of the cell of origin.

Lymphatic transport of exosomes as a rapid route of information dissemination to the lymph node

Scientific Reports

It is well documented that cells secrete exosomes, which can transfer biomolecules that impact recipient cells’ functionality in a variety of physiologic and disease processes. The role of lymphatic drainage and transport of exosomes is as yet unknown, although the lymphatics play critical roles in immunity and exosomes are in the ideal size-range for lymphatic transport. Through in vivo near-infrared (NIR) imaging we have shown that exosomes are rapidly transported within minutes from the periphery to the lymph node by lymphatics. Using an in vitro model of lymphatic uptake, we have shown that lymphatic endothelial cells actively enhanced lymphatic uptake and transport of exosomes to the luminal side of the vessel. Furthermore, we have demonstrated a differential distribution of exosomes in the draining lymph nodes that is dependent on the lymphatic flow. Lastly, through endpoint analysis of cellular distribution of exosomes in the node, we identified macrophages and B-cells as key players in exosome uptake. Together these results suggest that exosome transfer by lymphatic flow from the periphery to the lymph node could provide a mechanism for rapid exchange of infection-specific information that precedes the arrival of migrating cells, thus priming the node for a more effective immune response.

The relationship between lymphangion length and maximum pressure generation established through in vivo imaging and computational modeling

American Journal of Physiology

The intrinsic contraction of collecting lymphatic vessels serves as a pumping system to propel lymph against hydrostatic pressure gradients as it returns interstitial fluid to the venous circulation. We propose and validate that the maximum opposing outflow pressure along a chain of lymphangions at which flow can be achieved increases with the length of chain. Using minimally invasive near-infrared imaging to measure the effective pumping pressure at various locations in the rat tail, we demonstrate increases in the pumping pressure along the length of the tail. Computational simulations based on a microstructurally-motivated model of a chain of lymphangions informed from biaxial testing of isolated vessels was utilized to provide insight into the pumping mechanisms responsible for the pressure increases observed in vivo. These models suggest that the number of lympangions in the chain and smooth muscle cell force generation play a significant role in determining the maximum outflow pressure, while frequency of contraction has no effect. In vivo administration of nitric oxide (NO) attenuates lymphatic contraction, subsequently lowering the effective pumping pressure. Computational simulations suggest the reduction in contractile strength of smooth muscle cells in the presence of NO can account for the reductions in outflow pressure observed along the lymphangion chain in vivo. Thus combining computation modeling with multiple measurements of lymphatic pumping pressure provides a method for approximating intrinsic lymphatic muscle activity non-invasively in vivo, while also providing insight into factors that determine the extent that a lymphangion chain can transport fluid against an adverse pressure gradient.

Leukotriene B4 antagonism ameliorates experimental lymphedema

Science Translational Medicine

Acquired lymphedema is a cancer sequela and a global health problem currently lacking pharmacologic therapy. We have previously demonstrated that ketoprofen, an anti-inflammatory agent with dual 5-lipoxygenase and cyclooxygenase inhibitory properties, effectively reverses histopathology in experimental lymphedema. We show that the therapeutic benefit of ketoprofen is specifically attributable to its inhibition of the 5-lipoxygenase metabolite leukotriene B4 (LTB4). LTB4 antagonism reversed edema, improved lymphatic function, and restored lymphatic architecture in the murine tail model of lymphedema. In vitro, LTB4 was functionally bimodal: Lower LTB4 concentrations promoted human lymphatic endothelial cell sprouting and growth, but higher concentrations inhibited lymphangiogenesis and induced apoptosis. During lymphedema progression, lymphatic fluid LTB4 concentrations rose from initial prolymphangiogenic concentrations into an antilymphangiogenic range. LTB4 biosynthesis was similarly elevated in lymphedema patients. Low concentrations of LTB4 stimulated, whereas high concentrations of LTB4 inhibited, vascular endothelial growth factor receptor 3 and Notch pathways in cultured human lymphatic endothelial cells. Lymphatic-specific Notch1−/− mice were refractory to the beneficial effects of LTB4 antagonism, suggesting that LTB4 suppression of Notch signaling is an important mechanism in disease maintenance. In summary, we found that LTB4 was harmful to lymphatic repair at the concentrations observed in established disease. Our findings suggest that LTB4 is a promising drug target for the treatment of acquired lymphedema.

Post-prandial lymphatic pump function after a high-fat meal: a characterization of contractility, flow, and viscosity

American Journal of Physiology - Gastrointestinal and Liver Physiology

Dietary lipids are transported from the intestine through contractile lymphatics. Chronic lipid loads can adversely affect lymphatic function. However, the acute lymphatic pump response in the mesentery to a postprandial lipid meal has gone unexplored. In this study, we used the rat mesenteric collecting vessel as an in vivo model to quantify the effect of lipoproteins on vessel function. Lipid load was continuously monitored by using the intensity of a fluorescent fatty-acid analog, which we infused along with a fat emulsion through a duodenal cannula. The vessel contractility was simultaneously quantified. We demonstrated for the first time that collecting lymphatic vessels respond to an acute lipid load by reducing pump function. High lipid levels decreased contraction frequency and amplitude. We also showed a strong tonic response through a reduction in the end-diastolic and systolic diameters. We further characterized the changes in flow rate and viscosity and showed that both increase postprandially. In addition, shear-mediated Ca(2+) signaling in lymphatic endothelial cells differed when cultured with lipoproteins. Together these results show that the in vivo response could be both shear and lipid mediated and provide the first evidence that high postprandial lipid has an immediate negative effect on lymphatic function even in the acute setting.

Mechanotransduction activates Canonical Wnt/β-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves

Genes and Development

Lymphatic vasculature regulates fluid homeostasis by returning interstitial fluid to blood circulation. Lymphatic endothelial cells (LECs) are the building blocks of the entire lymphatic vasculature. LECs originate as a homogeneous population of cells predominantly from the embryonic veins and undergo stepwise morphogenesis to become the lymphatic capillaries, collecting vessels or valves. The molecular mechanisms underlying the morphogenesis of the lymphatic vasculature remain to be fully understood. Here we show that canonical Wnt/β-catenin signaling is necessary for lymphatic vascular morphogenesis. Lymphatic vascular-specific ablation of β-catenin in mice prevents the formation of lymphatic and lymphovenous valves. Additionally, lymphatic vessel patterning is defective in these mice, with abnormal recruitment of mural cells. We found that oscillatory shear stress (OSS), which promotes lymphatic vessel maturation, triggers Wnt/β-catenin signaling in LECs. In turn, Wnt/β-catenin signaling controls the expression of several molecules, including the lymphedema-associated transcription factor FOXC2. Importantly, FOXC2 completely rescues the lymphatic vessel patterning defects in mice lacking β-catenin. Thus, our work reveals that mechanical stimulation is a critical regulator of lymphatic vascular development via activation of Wnt/β-catenin signaling and, in turn, FOXC2.

Use of a novel portable three-dimensional scanner to measure limb volume and circumference in patients with filarial lymphedema

American Journal of Tropical Medicine and Hygeine

The World Health Organization’s Global Program to Eliminate Lymphatic Filariasis (LF) has reduced LF transmission worldwide, but millions remain affected by filarial lymphedema. Tools for clinically monitoring lymphedema in developing nations are limited. We tested a novel, portable, infrared three-dimensional (3D) scanner against water displacement (WD) and tape measurement of limb circumference (TMLC) among patients with filarial leg lymphedema in Galle, Sri Lanka. Outcomes were accuracy and reproducibility of scanner measurements. In parallel, we also tested the reproducibility of skin thickness ultrasound (STU) measurements. We examined 52 patients (104 limbs) with lymphedema of stages 0–6 (N = 28, 19, 20, 21, 2, 4, and 10, respectively). Scanner measurements correlated nearly perfectly with WD (r 2 = 0.9945) and TMLC values (r 2 > 0.9801). The average time required to acquire scanner measurements for both legs was 2.2 minutes, compared with 17.4, 7.5, and 31.7 minutes, respectively, for WD, TMLC, and STU. Median interexaminer coefficients of variation (CVs) for volume measurements were 1.1% (interquartile range [IQR] 0.5–2.1%) for WD and 1.7% (IQR 1.2–2.4%) for the 3D scanner. CVs for circumference measurements were 1.4% (IQR 0.8–2.4%) by TMLC and 1.3% (0.8–1.9%) by the 3D scanner. Median interexaminer CV for STU was 13.7% (IQR 8.5–21.3%). The portable 3D scanner noninvasively provided accurate and reproducible limb volume and circumference measurements in approximately 2 minutes per patient. This portable technology has the potential to greatly improve assessment and monitoring of lymphedema in the clinic and in the field.

Optimization of culture and analysis methods for enhancing long-term B. malayi survival, molting, and motility in vitro

Open Parasitology

Lymphatic filariasis is a neglected tropical disease caused by roundworm parasites such as Brugia malayi that spread via a mosquito vector. In vitro culture of these parasites provides controlled conditions to understand parasite biology and provides a cheaper way to screen potential micro- and macrofilaricides. Published studies have used a wide array of approaches and metrics regarding in vitro cultures of Brugia malayi; as a result, drawing comparisons and identifying the reasons why inability to reproduce outcomes are difficult. This study sought to determine conditions that ensure reproducible outcomes and used evaluation metrics that are easily measured and can be automated to ensure objectivity. We found culturing Brugia malayi L3 in endothelial basal media supplemented with 20% fetal bovine serum and 75 µM ascorbic acid in a temperature and humidity controlled incubator produced better survival and molting rates as well as longer and more motile parasites than previously reported. The benefit of ascorbic acid seemed to be unique to L3 parasites, as the addition of ascorbic acid to adult parasites had no significant impact on survival or motility. The methods reported in this study will help in designing experiments for both parasite behavior studies and drug screening applications for disease eradication.

Experimental models used to assess lymphatic contractile function

Lymphatic Research and Biology

TLR-exosomes exhibit distinct kinetics and effector function

Scientific Reports

The innate immune system is vital to rapidly responding to pathogens and Toll-like receptors (TLRs) are a critical component of this response. Nanovesicular exosomes play a role in immunity, but to date their exact contribution to the dissemination of the TLR response is unknown. Here we show that exosomes from TLR stimulated cells can largely recapitulate TLR activation in distal cells in vitro. We can abrogate the action-at-a-distance signaling of exosomes by UV irradiation, demonstrating that RNA is crucial for their effector function. We are the first to show that exosomes derived from poly(I:C) stimulated cells induce in vivo macrophage M1-like polarization within murine lymph nodes. These poly(I:C) exosomes demonstrate enhanced trafficking to the node and preferentially recruit neutrophils as compared to control exosomes. This work definitively establishes the differential effector function for exosomes in communicating the TLR activation state of the cell of origin.

Lymphatic transport of exosomes as a rapid route of information dissemination to the lymph node

Scientific Reports

It is well documented that cells secrete exosomes, which can transfer biomolecules that impact recipient cells’ functionality in a variety of physiologic and disease processes. The role of lymphatic drainage and transport of exosomes is as yet unknown, although the lymphatics play critical roles in immunity and exosomes are in the ideal size-range for lymphatic transport. Through in vivo near-infrared (NIR) imaging we have shown that exosomes are rapidly transported within minutes from the periphery to the lymph node by lymphatics. Using an in vitro model of lymphatic uptake, we have shown that lymphatic endothelial cells actively enhanced lymphatic uptake and transport of exosomes to the luminal side of the vessel. Furthermore, we have demonstrated a differential distribution of exosomes in the draining lymph nodes that is dependent on the lymphatic flow. Lastly, through endpoint analysis of cellular distribution of exosomes in the node, we identified macrophages and B-cells as key players in exosome uptake. Together these results suggest that exosome transfer by lymphatic flow from the periphery to the lymph node could provide a mechanism for rapid exchange of infection-specific information that precedes the arrival of migrating cells, thus priming the node for a more effective immune response.

A 2-D model of mechanically-mediated acute and long-term adaptations of contractility and geometry in lymphatics for characterization of lymphedema

Biomechanics and Modeling in Mechanobiology

A primary purpose of the lymphatic system is to transport fluid from peripheral tissues to the central venous system in order to maintain tissue–fluid balance. Failure to perform this task results in lymphedema marked by swelling of the affected limb as well as geometric remodeling and reduced contractility of the affected lymphatic vessels. The mechanical environment has been implicated in the regulation of lymphatic contractility, but it is unknown how changes in the mechanical environment are related to loss of contractile function and remodeling of the tissue. The purpose of this paper was to introduce a new theoretical framework for acute and long-term adaptations of lymphatic vessels to changes in mechanical loading. This theoretical framework combines a simplified version of a published lumped parameter model for lymphangion function and lymph transport, a published microstructurally motivated constitutive model for the active and passive mechanical behavior of isolated rat thoracic ducts, and novel models for acute mechanically mediated vasoreactive adaptations and long-term volumetric growth to simulate changes in muscle contractility and geometry of a single isolated rat thoracic duct in response to a sustained elevation in afterload. The illustrative examples highlight the potential role of the mechanical environment in the acute maintenance of contractility and long-term geometric remodeling, presumably aimed at meeting fluid flow demands while also maintaining mechanical homeostasis. Results demonstrate that contractility may adapt in response to shear stress to meet fluid flow demands and show that pressure-induced long-term geometric remodeling may attenuate these adaptations and reduce fluid flow. The modeling framework and illustrative simulations help suggest relevant experiments that are necessary to accurately quantify and predict the acute and long-term adaptations of lymphangions to altered mechanical loading.