Cation Channelopathies: Novel Insights Into Generalized Lymphatic Dysplasia

The Lymphatic vasculature, a network of conduits first described in the 17th century¹, primarily functions parallel to the blood vascular system to efficiently drain excess fluid from the tissue interstitium. In addition, the lymphatic vascular system regulates dietary lipid transport, immune responses, clearance of cerebrospinal fluid, and aqueous humor drainage². Beyond Gaspare Aselli’s anatomical description of lymphatic vessels in 1622, modern biomedical research has identified the molecular framework underlying lymphatic endothelial cell structure and function in development and disease^{3, 4}. In the capillary vascular bed, arterial and venous pressure gradient differences generate net fluid flux out of circulation (Ernest Starling’s equation) into the interstitium leading to interstitial fluid (lymph) accumulation. Lymphatic capillaries collect excess interstitial fluid and transport it to the subclavian vein via the thoracic duct⁴. Developmental lymphatic malformations or structural/functional lymphatic defects secondary to diseases cause aberrant accumulation of interstitial fluid, leading to primary or secondary lymphedema³.

Approximately 28 genes are causally linked to primary lymphedema, including transcription factors SOX18, FOXC2, GATA2, growth factor-RTK signaling pathway components VEGFC, VEGFR3, ADAMTS3, CCBE1, membrane proteins GJC2, FAT4, CELSR1, and mechanosensory protein PIEZO1². Indeed, studies over the last decade have identified mechanical force as a critical regulator of lymphatic vessel development, specifically collecting lymphatic vessel maturation and the development of lymphatic valves required for a unidirectional flow of lymph^{5, 6}. PIEZO1, and its homolog PIEZO2, were initially identified as a mechanically activated cation channel, which allows the flow of both Na⁺ and Ca⁺⁺ ions in neuronal cell populations⁷. Genetic loss-of-function studies in mice determined that blood flow-mediated shear stress activates Piezo1 to maintain endothelial cell alignment in the aorta⁸. Similarly, endothelial Peizo1 increases the production of eNOS, an established vasodilator, to promote shear stress-induced vasodilation in adult mice⁸. In this issue of Circulation Research, Choi D et al.⁹ identify Piezo1 as a critical link between fluid-flow generated shear stress and intracellular calcium influx within lymphatic endothelial cells. Aberrant intracellular calcium influx inhibits the sprouting of lymphatic vessels, leading to generalized lymphatic dysplasia, a rare syndrome characterized by chylothorax, chylous ascites, and lymphedema⁹.

Patients with generalized lymphatic dysplasia often exhibit biallelic loss-of-function PIEZO1 mutations, including splice site, indel/missense, and copy number variants (Table 1). Immunoblots of red blood cells (RBCs) isolated from patients show that PIEZO1 protein expression is severely reduced even in cases harboring missense mutations, suggesting that protein stability is adversely affected¹⁰. Human genetic data support a causal role of PIEZO1 loss-of-function in generalized lymphatic dysplasia^{10, 11}. Patients with gain-of-function mutations in PIEZO1 exhibit dehydrated hereditary stomatocytosis with or without pseudo-hyperkalemia and/or perinatal edema, a dominantly inherited hemolytic disorder¹². However, the etiopathology of the two PIEZO1-related diseases is not predicted to arise from a common mechanism, although fetal/neonatal edema is a shared clinical feature. RBCs have a stomatocyte appearance in dehydrated hereditary stomatocytosis, and ektacytometry curves are left-shifted, indicating RBC dehydration. One of the proposed mechanisms of PIEZO1 gain-of-function in dehydrated hereditary stomatocytosis is delayed inactivation of the PIEZO1 channels through missense change of highly conserved residues, leading to elevated calcium influx¹². This over-activation of PIEZO1 may induce pleiotropic effects contributing to anemia, including dehydration via potassium efflux, RBC membrane deformation, and altered iron metabolism in hepatocytes. In addition, PIEZO1 expression in fetal peritoneal lymphatics and its role in endothelial calcium influx suggest a lymphatic component to dehydrated hereditary stomatocytosis, although further work is needed to establish this association¹³.

Table 1:

PIEZO1 variants in patients with generalized lymphatic dysplasia.

Type of variation	nucleotide variant	Amino acid variant	Lymphatic Phenotype in Patients
Truncation	c.6682C>T	Q2228stop	Pleural effusion, ascites, skin edema, polyhydramnios, lymphedema, chylothorax, pectus excavatum, genital edema, splenomegaly, periorbital edema10
Truncation	c.3206G>A	W1069stop	Bilateral pleural effusions, scalp, facial and abdominal wall edema, trace pericardial effusion and marked polyhydramnios
Truncation	c.3373C>T	Q1125stop	Lower limb Lymphedema, Facial and upper Limb Lymphedema in some individuals
Truncation	c. 2263G>T	E755stop	Pleural effusion, ascites, skin edema, polyhydramnios, lymphedema10
Truncation	c.4888G>T	E1630stop	Postnatal lymphedema, chylothorax, pleural effusion, recurrent cellulitis in face and lower limbs, recurrent facial cellulitis, genital edema10
Single nucleotide variant	c.7366C>T	R2456C	Pleural effusion, ascites, skin edema, polyhydramnios, generalized edema at birth, chylothorax, periorbital edema10
Single nucleotide variant	c.2815C>A	L939M	Pleural effusion, ascites, skin edema, polyhydramnios, generalized edema at birth, chylothorax, periorbital edema10
Single nucleotide variant	c.7374C>G	F2458L	Pleural effusion, ascites, skin edema, polyhydramnios, generalized edema at birth, chylothorax, periorbital edema10
Single nucleotide variant	c.7289C>T	P2430L	Pleural effusion, polyhydramnios, neonatal edema, lymphedema, epicanthic folds, cellulitis10
Single nucleotide variant	c.6085G>C	G2029R	Hydrops, bilateral chylothorax, lymphedema, chronic pleural effusion, tachypnoea mild infraorbital hypoplasia with subjective hypertelorism, slightly flat facial gestalt with mild periorbital edema11
Single nucleotide variant	c.6511G>T	V2171F	Pleural effusion, ascites, neonatal edema, lymphedema, gastroesophageal reflux, intestinal lymphangiectasia10
Single nucleotide variant	c.4072C>T	R1358C	Upper limb Lymphedema
Single nucleotide variant	c.5033C>T	A1678V	Upper limb Lymphedema
Single nucleotide variant	c.2858G>A	R953H	Pleural effusion, Lower limb Lymphedema, hydrocele
Single nucleotide variant	c.5162C>G	S1721W	Subcutaneous edema along the head, bilateral pleural effusion
Single nucleotide variant	c.7316G>A	G2439D	Lower limb Lymphedema, Facial and upper Limb Lymphedema in some individuals
Single nucleotide variant	c.1792G>C	V598L	Perinatal edema, later in life bilateral lower limb lymphedema
Single nucleotide variant	c.6208A>C	K2070Q	Bilateral pleural effusions, scalp, facial and abdominal wall edema, trace pericardial effusion and marked polyhydramnios
Deletion	c.5725delA	R1909Efs*12	Non-immune hydrops fetalis, congenital lymphatic dysplasia, fetal pleural effusion, peripheral edema, hydrocele, Lower limb Lymphedema
Deletion	chr16:88,782,477–88,876,207	exon 1–50	Pleural effusion, Lower limb Lymphedema, hydrocele
Donor splice variant	c.3455+1G>A	S1153Wfs21*	Hydrops and bilateral chylothorax at birth, persistent lymphoedema of legs, torso and face, chronic pleural effusions11
Donor splice variant	c.3796+1G>A	Exon 26 skipped	All 4 limbs and facial lymphedema, intestinal lymphangiectasia10
Donor splice variant	c.1669+1G>A	27 codons from Intron 13 included	Bilateral Lower limb and facial lymphedema10
Splice variant	c.6165–7G>A	Intron 42	Non-immune hydrops fetalis, congenital lymphatic dysplasia, fetal pleural effusion, peripheral edema, hydrocele, Lower limb Lymphedema

Initial studies in mice and human lymphatic endothelial cells showed that Piezo1 loss-of-function affects lymphatic valve development and maintenance^{14, 15}. The lymphatic system relies on intraluminal valves to ensure a unidirectional flow of lymph, and valve dysfunction leads to disrupted fluid homeostasis and lymphatic disease³. In vitro experiments on cultured human lymphatic endothelial cells revealed that PIEZO1 is required for oscillatory shear stress-mediated upregulation of lymphatic valve-specific genes, including FOXC2, GATA2, CX37, and LAMA5¹⁵. These studies showed defective or missing lymphatic valves as a primary mechanism for Piezo1-related generalized lymphatic dysplasia^{14, 15}. Choi D et al. show through a series of elegant experiments that Piezo1 plays a role in lymphangiogenic sprouting and vessel maintenance, mediated by Orai1, a calcium release-activated calcium channel component⁹. Choi D et al. reveal that Piezo1 acts upstream of Orai1 to promote the flow-induced gene expression of Dtx1 and Dtx3L, downregulating Notch activity and inducing lymphatic sprouting⁹. Remarkably, lymphatic Piezo1 conditional deletion in mice recapitulates sprouting lymphatic vessel defects observed in lymphatic Orai1 or Klf2 knockout murine embryos and lymphatic Notch1 gain-of-function murine embryos⁹. While defective lymphatic sprouting or valve morphogenesis is not mutually exclusive, it has been suggested that a failure in the initial lymphatics and not the collectors are the culprit in generalized lymphatic dysplasia. Further work detailing the relationship between sprouting lymphatic vessels and valve function will be necessary to understand the etiopathogenesis of generalized lymphatic dysplasia.

Choi D et al. identify Piezo1 and Orai1 as promising candidates for pre-clinical studies on PIEZO1-related generalized lymphatic dysplasia⁹. Yoda1, the first discovered chemical agonist of Piezo1, was identified in a screen of ~3.25 million small molecules¹⁶. Yoda1 acts as a molecular wedge, lowering the activation threshold of Piezo by de-coupling Repeat A and the N-terminus of the arm¹⁶. Choi D et al. show that Yoda1 administration can improve lymphatic function and induce lymphatic sprouting in a mouse model of surgery-associated lymphedema⁹. Choi D et al. also demonstrate that the Orai1 activator, IA65, promotes lymphatic growth and could be a candidate for pre-clinical animal model testing⁹. In addition to Yoda1, Jedi1/2 are two additional agonists discovered in a screen of 3000 compounds for targets that could evoke Ca⁺⁺ release in response to mouse PIEZO1¹⁷. Overall, Choi D et al.⁹ identify promising compounds to treat both primary and secondary lymphedema.