TRPV4-Expressing Tissue-Resident Macrophages Regulate the Function of Collecting Lymphatic Vessels via Thromboxane A2 Receptors in Lymphatic Muscle Cells.

Rationale: TRPV4 channels are critical regulators of blood vascular function and have been shown to be dysregulated in many disease conditions in association with inflammation and tissue fibrosis. These are key features in the pathophysiology of lymphatic system diseases, including lymphedema and lipedema; however, the role of TRPV4 channels in the lymphatic system remains largely unexplored. TRPV4 channels are calcium permeable, non-selective cation channels that are activated by diverse stimuli, including shear stress, stretch, temperature, and cell metabolites, which may regulate lymphatic contractile function. Objective: To characterize the expression of TRPV4 channels in collecting lymphatic vessels and to determine the extent to which these channels regulate the contractile function of lymphatics. Methods and Results: Pressure myography on intact, isolated, and cannulated lymphatic vessels showed that pharmacological activation of TRPV4 channels with GSK1016790A (GSK101) led to contractile dysregulation. The response to GSK101 was multiphasic and included, 1) initial robust constriction that was sustained for ≥1 minute and in some instances remained for ≥4 minutes; and 2) subsequent vasodilation and partial or complete inhibition of lymphatic contractions associated with release of nitric oxide. The functional response to activation of TRPV4 channels displayed differences across lymphatics from four anatomical regions, but these differences were consistent across different species (mouse, rat, and non-human primate). Importantly, similar responses were observed following activation of TRPV4 channels in arterioles. The initial and sustained constriction was prevented with the COX inhibitor, indomethacin. We generated a controlled and spatially defined single-cell RNA sequencing (scRNAseq) dataset from intact and microdissected collecting lymphatic vessels. Our data uncovered a subset of macrophages displaying the highest expression of Trpv4 compared to other cell types within and surrounding the lymphatic vessel wall. These macrophages displayed a transcriptomic profile consistent with that of tissue-resident macrophages (TRMs), including differential expression of Lyve1 , Cd163 , Folr2 , Mrc1 , Ccl8 , Apoe , Cd209f , Cd209d , and Cd209g ; and at least half of these macrophages also expressed Timd4. This subset of macrophages also highly expressed Txa2s , which encodes the thromboxane A2 (TXA2) synthase. Inhibition of TXA2 receptors (TXA2Rs) prevented TRPV4-mediated contractile dysregulation. TXA2R activation on LMCs caused an increase in mobilization of calcium from intracellular stores through Ip3 receptors which promoted store operated calcium entry and vasoconstriction. Conclusions: Clinical studies have linked cancer-related lymphedema with an increased infiltration of macrophages. While these macrophages have known anti-inflammatory and pro-lymphangiogenic roles, as well as promote tissue repair, our results point to detrimental effects to the pumping capacity of collecting lymphatic vessels mediated by activation of TRPV4 channels in macrophages. Pharmacological targeting of TRPV4 channels in LYVE1-expressing macrophages or pharmacological targeting of TXA2Rs may offer novel therapeutic strategies to improve lymphatic pumping function and lymph transport in lymphedema.

Right ventricular preload and afterload challenge induces contractile dysfunction and arrhythmia in isolated hearts of dystrophin-deficient male mice.

Duchenne muscular dystrophy (DMD) is an X-linked recessive myopathy due to mutations in the dystrophin gene. Diaphragmatic weakness in DMD causes hypoventilation and elevated afterload on the right ventricle (RV). Thus, RV dysfunction in DMD develops early in disease progression. Herein, we deliver a 30-min sustained RV preload/afterload challenge to isolated hearts of wild-type (Wt) and dystrophic (Dmdmdx-4Cv) mice at both young (2-6 month) and middle-age (8-12 month) to test the hypothesis that the dystrophic RV is susceptible to dysfunction with elevated load. Young dystrophic hearts exhibited greater pressure development than wild type under baseline (Langendorff) conditions, but following RV challenge exhibited similar contractile function as wild type. Following the RV challenge, young dystrophic hearts had an increased incidence of premature ventricular contractions (PVCs) compared to wild type. Hearts of middle-aged wild-type and dystrophic mice had similar contractile function during baseline conditions. After RV challenge, hearts of middle-aged dystrophic mice had severe RV dysfunction and arrhythmias, including ventricular tachycardia. Following the RV load challenge, dystrophic hearts had greater lactate dehydrogenase (LDH) release than wild-type mice indicative of damage. Our data indicate age-dependent changes in RV function with load in dystrophin deficiency, highlighting the need to avoid sustained RV load to forestall dysfunction and arrhythmia.

Advanced age and female sex protect cerebral arteries from mitochondrial depolarization and apoptosis during acute oxidative stress.

Aging increases reactive oxygen species (ROS) which can impair vascular function and contribute to brain injury. However, aging can also promote resilience to acute oxidative stress. Therefore, we tested the hypothesis that advanced age protects smooth muscle cells (SMCs) and endothelial cells (ECs) of posterior cerebral arteries (PCAs; diameter, ∼80 µm) during exposure to H2O2. PCAs from young (4-6 months) and old (20-26 months) male and female C57BL/6 mice were isolated and pressurized (~70 mm Hg) to evaluate cell death, mitochondrial membrane potential (ΔΨm), ROS production, and [Ca2+]i in response to H2O2 (200 µM, 50 min). SMC death and ΔΨm depolarization were greater in PCAs from males vs. females. Aging increased ROS in PCAs from both sexes but increased SMC resilience to death only in males. Inhibiting TRPV4 channels with HC-067047 (1 µM) or Src kinases with SU6656 (10 µM) reduced Ca2+ entry and SMC death to H2O2 most effectively in PCAs from young males. Activating TRPV4 channels with GSK1016790A (50 nM) evoked greater Ca2+ influx in SMCs and ECs of PCAs from young vs. old mice but did not induce cell death. However, when combined with H2O2, TRPV4 activation exacerbated EC death. Activating Src kinases with spermidine (100 µM) increased Ca2+ influx in PCAs from males vs. females with minimal cell death. We conclude that in males, chronic oxidative stress during aging increases the resilience of cerebral arteries, which contrasts with inherent protection in females. Findings implicate TRP channels and Src kinases as targets to limit vascular damage to acute oxidative injury.