We previously reported increased renal lymphatic density in multiple mouse models of hypertension, and further augmenting renal lymphatics lowers blood pressure. However, whether interstitial levels of hypertensive stimuli have a direct effect on lymphatics or an indirect effect through secreted immune cell factors has not been examined. We hypothesized that hypertensive stimuli directly increases lymphatic endothelial cell (LEC) proliferation and increases sprouting of mouse mesometrial lymphatic vessels. Murine LECs were cultured and treated with angiotensin II (angII), salt, and asymmetric dimethylarginine (ADMA) for 24 hours. To mimic the
in vivoenvironment, a lymphatic-specific reporter mouse (Prox1-tdTomato) mesometrium tissue explant was treated with either the same hypertensive stimuli or with hypertensive conditioned media for 8 days. Mesometrial vascular beds were cultured in DMEM supplemented with 20% fetal bovine serum to induce lymphatic sprouting and this was replenished every day. The conditioned media was made by treating murine splenocytes for 24 hours with the same hypertensive stimuli. These stimuli had no effect on murine LEC proliferation. Hypertensive stimuli significantly decreased mesometrial lymphatic vessel sprout length (SL) and sprout number (SN) compared to controls (control SL in pixels by ImageJ analysis: 34.0 2.6, angII: 3.7 2.6, salt: 2.67 2.18, ADMA: 9.06 5.12, all p<0.05; control SN: 7 3, angII: 0 0, salt: 0 0, ADMA: 1 1, all p<0.05). Conditioned media treatment normalized SL and SN by day 8 for all hypertensive stimuli except salt. In conclusion, hypertensive stimuli directly inhibit mesometrial lymphangiogenesis, but this was mitigated by hypertensive stimuli induced immune cell secreted factors.