Vitamin E Attenuates Red-Light-Mediated Vasodilation: The Benefits of a Mild Oxidative Stress.

Red light (670 nm) energy controls vasodilation via the formation of a transferable endothelium-derived nitric oxide (NO)-precursor-containing substance, its intracellular traffic, and exocytosis. Here we investigated the underlying mechanistic effect of oxidative stress on light-mediated vasodilation by using pressure myography on dissected murine arteries and immunofluorescence on endothelial cells. Treatment with antioxidants Trolox and catalase decreased vessel dilation. In the presence of catalase, a lower number of exosomes were detected in the vessel bath. Light exposure resulted in increased cellular free radical levels. Mitochondrial reactive oxygen species were also more abundant but did not alter cellular ATP production. Red light enhanced the co-localization of late exosome marker CD63 and cellular S-nitrosoprotein to a greater extent than high glucose, suggesting that a mild oxidative stress favors the localization of NO precursor in late exosomes. Exocytosis regulating protein Rab11 was more abundant after irradiation. Our findings conclude that red-light-induced gentle oxidative stress facilitates the dilation of blood vessels, most likely through empowering the traffic of vasodilatory substances. Application of antioxidants disfavors this mechanism.

Red light mediates the exocytosis of vasodilatory vesicles from cultured endothelial cells: a cellular, and ex vivo murine model.

We have previously established that 670 nm energy induces relaxation of blood vessels via an endothelium derived S-nitrosothiol (RSNO) suggested to be embedded in vesicles. Here, we confirm that red light facilitates the exocytosis of this vasodilator from cultured endothelial cells and increases ex vivo blood vessel diameter. Ex vivo pressurized and pre-constricted facial arteries from C57Bl6/J mice relaxed 14.7% of maximum diameter when immersed in the medium removed from red-light exposed Bovine Aortic Endothelial Cells. In parallel experiments, 0.49 nM RSNO equivalent species was measured in the medium over the irradiated cells vs dark control. Electron microscopy of light exposed endothelium revealed significant increases in the size of the Multi Vesicular Body (MVB), a regulator of exosome trafficking, while RSNO accumulated in the MVBs as detected with immunogold labeling electron microscopy (1.8-fold of control). Moreover, red light enhanced the presence of F-actin related stress fibers (necessary for exocytosis), and the endothelial specific marker VE-cadherin levels suggesting an endothelial origin of the extracellular vesicles. Flow cytometry coupled with DAF staining, an indirect sensor of nitric oxide (NO), indicated significant amounts of NO within the extracellular vesicles (1.4-fold increase relative to dark control). Therefore, we further define the mechanism on the 670 nm light mediated traffic of endothelial vasodilatory vesicles and plan to leverage this insight into the delivery of red-light therapies.

Red Light Mitigates the Deteriorating Placental Extracellular Matrix in Late Onset of Preeclampsia and Improves the Trophoblast Behavior.

Preeclampsia is a serious pregnancy disorder which in extreme cases may lead to maternal and fetal injury or death. Preexisting conditions which increase oxidative stress, e.g., hypertension and diabetes, increase the mother's risk to develop preeclampsia. Previously, we established that when the extracellular matrix is exposed to oxidative stress, trophoblast function is impaired, and this may lead to improper placentation. We investigated how the oxidative ECM present in preeclampsia alters the behavior of first trimester extravillous trophoblasts. We demonstrate elevated levels of advanced glycation end products (AGE) and lipid oxidation end product 4-hydroxynonenal in preeclamptic ECM (28%, and 32% increase vs control, respectively) accompanied with 35% and 82% more 3-chlorotyrosine and 3-nitrotyrosine vs control, respectively. Furthermore, we hypothesized that 670 nm phototherapy, which has antioxidant properties, reverses the observed trophoblast dysfunction as depicted in the improved migration and reduction in apoptosis. Since NO is critical for placentation, we examined eNOS activity in preeclamptic placentas compared to healthy ones and found no differences; however, 670 nm light treatment triggered enhanced NO availability presumably by using alternative NO sources. Light exposure decreased apoptosis and restored trophoblast migration to levels in trophoblasts cultured on preeclamptic ECM. Moreover, 670 nm irradiation restored expression of Transforming Growth Factor (TGFß) and Placental Growth Factor (PLGF) to levels observed in trophoblasts cultured on healthy placental ECM. We conclude the application of 670 nm light can successfully mitigate the damaged placental microenvironment of late onset preeclampsia as depicted by the restored trophoblast behavior.

In Vivo Characterization of a Red Light-Activated Vasodilation: A Photobiomodulation Study.

Nitric oxide dependent vasodilation is an effective mechanism for restoring blood flow to ischemic tissues. Previously, we established an ex vivo murine model whereby red light (670 nm) facilitates vasodilation via an endothelium derived vasoactive species which contains a functional group that can be reduced to nitric oxide. In the present study we investigated this vasodilator in vivo by measuring blood flow with Laser Doppler Perfusion imaging in mice. The vasodilatory nitric oxide precursor was analyzed in plasma and muscle with triiodide-dependent chemiluminescence. First, a 5-10 min irradiation of a 3 cm2 area in the hind limb at 670 nm (50 mW/cm2) produced optimal vasodilation. The nitric oxide precursor in the irradiated quadriceps tissue decreased significantly from 123 ± 18 pmol/g tissue by both intensity and duration of light treatment to an average of 90 ± 17 pmol/g tissue, while stayed steady (137 ± 21 pmol/g tissue) in unexposed control hindlimb. Second, the blood flow remained elevated 30 min after termination of the light exposure. The nitric oxide precursor content significantly increased by 50% by irradiation then depleted in plasma, while remained stable in the hindlimb muscle. Third, to mimic human peripheral artery disease, an ameroid constrictor was inserted on the proximal femoral artery of mice and caused a significant reduction of flow. Repeated light treatment for 14 days achieved steady and significant increase of perfusion in the constricted limb. Our results strongly support 670 nm light can regulate dilation of conduit vessel by releasing a vasoactive nitric oxide precursor species and may offer a simple home-based therapy in the future to individuals with impaired blood flow in the leg.