Rhythmic Contractions of Lymph Vessels and Lymph Flow Are Disrupted in Hypertensive Rats

Abstract

Background: Hypertension increases the risk of lymphedema in patients with comorbidities, but whether hypertension directly compromises lymph vessel (LV) function and lymph flow is unclear. We compared the contractions of mesenteric LVs ex vivo and lymph flow in vivo between normotensive and Ang II (angiotensin II)-induced hypertensive rats and explored the ionic basis of contractile patterns. Key studies were recapitulated in spontaneously hypertensive rats and control Wistar-Kyoto rats.

Methods: Video microscopy continuously recorded the diameters of cannulated rat mesenteric LVs, and high-speed optical imaging estimated mesenteric lymph flow in vivo. Jess capillary Western electrophoresis evaluated expression levels of ion channel proteins.

Results: Isolated LVs from Ang II-induced hypertensive rats exhibited dysrhythmic contractions, whereas LVs from both Ang II-induced hypertensive rats and spontaneously hypertensive rats exhibited reduced diastolic diameters and cross-sectional flow. Mesenteric lymph flow in vivo was 2.9-fold lower in Ang II-induced hypertensive rats compared with normotensive rats. Surprisingly, the LVs from Ang II-induced hypertensive rats expressed fewer intact L-type Ca²⁺ channel pore proteins and more modulatory cleaved C-terminal fragments. However, pharmacological block of voltage-gated K⁺ channels but not other K⁺ channel types in control LVs established the pattern of contractile dysfunction observed in hypertension. Jess capillary Western electrophoresis analysis confirmed a loss of Shaker-type K_V1.2 channels in LVs from hypertensive rats.

Conclusions: We provide initial evidence of lymphatic contractile dysfunction and compromised lymph flow in hypertensive rats, which may be caused by a loss of K_V1.2 channels in the lymphatic muscle cells.

Keywords: hypertension; lymphatic vessels; lymphedema; muscle cells; muscle contraction; rats.

Figure 1.

Recording of dysrhythmic contractions in cannulated mesenteric lymph vessels (LVs) of angiotensin II-induced hypertensive (AHT) rats. A, Representative diameter traces show (left) rhythmic contractions of normotensive (NT) LVs and (right) dysrhythmic contractions of AHT LVs. B, Manhattan plot revealed a more variable contraction interval in AHT LVs, leading to an increased coefficient of variance of the interval compared with NT LVs. AHT LVs exhibited a trend toward increased Ca²⁺–dependent tone, evident as a greater dilator response to (C) nifedipine (NIF) and Ca²⁺-free physiological salt solution (PSS) compared with NT LVs. D, Passive LV diameter was not significantly different between NT and AHT LVs. E, Calculated cross-sectional flow was markedly lower in AHT compared with NT LVs and was associated with (F) reduced end-diastolic diameter. F, End-systolic diameter and (F) contraction frequency were not significantly different between NT and AHT LVs (n=6–8 per group; **P<0.01 and *P<0.05).

Figure 2.

In vivo mesenteric lymph flow is decreased in angiotensin II-induced hypertensive (AHT) rats. A, Basal in vivo positive volumetric lymph flow was significantly lower in lymph vessels (LVs) of AHT compared with normotensive (NT) rats. B, Cell tracking in LVs in vivo, in which upward deflections reflect average positive (distal-to-proximal) cell velocities in (i) NT LVs and (ii) AHT LVs. Downward deflections reflect average negative velocity caused by LV valve closure. C, Representative still images of LVs of (i) NT and (ii) AHT rats used for frame-by-frame cell tracking (300-frames per second video). Blue and red arrows point to individual cells in lymph fluid. D, Positive volumetric lymph flow in vivo was reduced significantly more in AHT LVs compared with NT LVs after superfusion with Ca²⁺-free physiological salt solution (PSS; n=7–8 per group; **P<0.01 and *P<0.05).

Figure 3.

Comparison of L-type Ca2+ channel protein between whole-vessel lysates from normotensive and angiotensin II-induced hypertensive (AHT) lymph vessels (LVs). A, Jess capillary Western electrophoresis revealed lower expression of the intact α_1C pore protein in AHT compared with normotensive (NT) LVs (top) and increased expression of the cleaved C-terminal (CCt) fragment (middle). Expression of Na⁺-K⁺-ATPase was used as a loading control (bottom). Average fold change after normalization using the loading control for each lane confirms (B) decreased intact α_1C protein and (C) increased CCt in AHT LVs compared with NT LVs (n=5 per group; *P<0.05).

Figure 4.

Pharmacological block of delayed rectifier K_V channels induces dysrhythmic contractions and increased Ca²⁺–dependent tone in normotensive lymph vessels (LVs). Addition of (A) barium chloride (BaCl₂; 15 µmol/L), (B) glibenclamide (Gbc; 15 µmol/L), or (C) iberiotoxin (Ibtx; 1 µmol/L) to block K_IR, K_ATP, or BK channels, respectively, did not significantly alter rhythmic contractions or end-diastolic diameter in normotensive (NT) LVs (n=3). D, In contrast, the addition of 500-µmol/L 4-aminopyridine (4-AP) to block delayed rectifier K_V channels triggered dysrhythmic contractions and decreased diastolic diameter. Dysrhythmic contractions were eliminated, and diastolic diameter was restored by subsequent superfusion with Ca²⁺-free physiological salt solution (PSS). E, 4-AP increased Ca²⁺–dependent tone that was evident as a reduced diastolic diameter in NT LVs (n=4). F, Jess capillary Western electrophoresis revealed a marked downregulation of the delayed rectifier K_V1.2 subtype (≈80 kDa) in LVs of angiotensin II-induced hypertensive (AHT) compared with NT rats. Data presented as average fold change after normalization to the loading control (n=5 per group; *P<0.05).

Figure 5.

Diameter recordings of spontaneous contractions in cannulated mesenteric lymph vessels (LVs) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). A, Sample recordings of LVs from 3 WKY rats revealed rhythmic contractions (left and middle recordings) or dysrhythmic contractions and minimal Ca²⁺-dependent basal tone. B, A sampling of 3 LVs from SHR exhibited a pattern of reduced diastolic and systolic diameter and highly variable Ca²⁺-dependent basal tone. NIF indicates nifedipine; and PSS, physiological salt solution.

Figure 6.

Contractile parameters and Kv1.2 protein expression in mesenteric lymph vessels (LVs) of Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). A, Manhattan plot revealed no significant difference in contraction interval between WKY and SHR LVs, resulting in similar coefficients of variation. Basal Ca²⁺–dependent tone also was not significantly different between WKY and SHR LVs, as evidenced by similar dilator responses to (B) nifedipine (NIF) and Ca²⁺-free physiological salt solution (PSS). C, Passive diameter in Ca²⁺-free PSS was smaller in LVs of SHR compared with WKY rats. D, Calculated cross-sectional flow also was markedly lower in SHR LVs and was associated with (E) reduced end-diastolic diameter and (E) reduced end-systolic diameter. E, Contraction frequency was not significantly different between WKY and SHR LVs (n=9–10 per group; *P<0.0). F, Jess capillary Western electrophoresis revealed a marked downregulation of the delayed rectifier K_V1.2 subtype in LVs of SHR compared with WKY rats. Average fold change after normalization to the loading control in each lane confirmed decreased K_V1.2 protein in SHR LVs compared with WKY LVs (n=4 per group; **P<0.01).