Lymph transport relies critically on the intrinsic contractions of lymphatic smooth muscle (SM) cells in the walls of collecting lymphatic vessels to propel lymph against a hydrostatic pressure gradient. These contractions must be coordinated within each pumping unit, the lymphangion, to efficiently propel lymph downstream to the next segment. Synchronized contractions depend on tight electrical coupling of lymphatic SMCs through connexins (Cx) - gap junction proteins that link adjacent cells and allow the transfer of electrical current. The significance of understanding how contractions are coordinated is underscored by studies suggesting that impaired or uncoordinated lymphatic vessel contractions contribute to both congenital and acquired forms of lymphedema. Notably, women with Cx47 mutations have a higher incidence of breast cancer-related lymphedema of the extremities and patients with Cx47 or Cx43 mutations develop primary lymphedema, reputedly due to a contraction coordination deficit in collecting lymphatic vessels. Little is know about Cx expression and electrical coupling between the cells of the lymphatic wall. A better understanding of how electrical signals are conducted and coordinated within and along the lymphatic wall requires knowledge of the specific Cx isoforms expressed in the SM and endothelial cell (EC) layers as well as direct measures of electrical coupling between the cells. We have recently developed a preparation ideally suited to address these issues: the isolated popliteal lymphatic vessel of the mouse. We can control pressure/flow, measure membrane potential in both SMCs and ECs, and quantitatively assess the degree of cell-cell coupling by passing current between two recording microelectrodes at defined separations. Our preliminary data suggest 1) lymphatic vessels show EC-specific expression of Cx37 and Cx43 but SM-specific expression of Cx45; 2) there is robust EC-EC electrical coupling, good SM-SM coupling, but relatively poor SM-EC coupling. This is in stark contrast to what is found in arterioles. Our central hypothesis is that the lymphatic wall exhibits a high degree of SM-SM electrical coupling between, but not across, valves to allow focal generation, conduction and synchronization of action potentials (APs) within lymphangions; further, we hypothesize that the SM layer is essentially uncoupled electrically from the EC layer, due to poor SM-EC Cx expression, and is optimized to conduct a depolarizing wave rather than a hyperpolarization wave. This hypothesis will be tested by 3 specific aims: 1) Determine the extent of electrical and Cx coupling between SM and EC cells in the lymphatic wall; 2) Determine which cells of the lymphatic wall initiate electrical pacemaking; 3) Determine the factors controlling the direction and conduction of lymphatic pacemaker waves. We will take advantage of GFP reporter mice and global and tissue-specific knock-outs of specific Cx isoforms. Accomplishment of these aims will advance our understanding of how lymphatic contractions are coordinated, enabling potential pharmacological rescue of the dysfunctional lymphatic pump in lymphedema.

Public Health Relevance

Lymphedema affects over 10 million people annually in the USA, yet little is known about how and why lymphatic vessels become dysfunctional in lymphedema. Synchronized contractions of smooth muscle cells in the lymphatic vessel wall are of critical importance to lymphatic vessel contraction and lymph pumping: this project focuses on junctional proteins, the connexins (Cx), that link those cells to allow cell-cell communication. The significance of understanding how lymphatic vessel contractions are coordinated is underscored by recent genetic studies showing that women with Cx47 mutations have a higher incidence of breast cancer-related lymphedema and patients with Cx47 or Cx43 mutations develop primary lymphedema, reputedly due to a contraction coordination deficit in collecting lymphatic vessels.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL125608-04
Application #
9393354
Study Section
Hypertension and Microcirculation Study Section (HM)
Program Officer
Tolunay, Eser
Project Start
2014-12-24
Project End
2018-11-30
Budget Start
2017-12-01
Budget End
2018-11-30
Support Year
4
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Missouri-Columbia
Department
Pharmacology
Type
Schools of Medicine
DUNS #
153890272
City
Columbia
State
MO
Country
United States
Zip Code
65211
Munger, Stephanie J; Davis, Michael J; Simon, Alexander M (2017) Defective lymphatic valve development and chylothorax in mice with a lymphatic-specific deletion of Connexin43. Dev Biol 421:204-218
Jamalian, Samira; Davis, Michael J; Zawieja, David C et al. (2016) Network Scale Modeling of Lymph Transport and Its Effective Pumping Parameters. PLoS One 11:e0148384
Scallan, Joshua P; Zawieja, Scott D; Castorena-Gonzalez, Jorge A et al. (2016) Lymphatic pumping: mechanics, mechanisms and malfunction. J Physiol 594:5749-5768
Chakraborty, Sanjukta; Davis, Michael J; Muthuchamy, Mariappan (2015) Emerging trends in the pathophysiology of lymphatic contractile function. Semin Cell Dev Biol 38:55-66