The proper physiological function of tissues relies on the timely and appropriate delivery of blood flow. Disruption or derangement of blood flow, either through physical obstruction or impaired responses to cellular agonists, results in ischemia and end organ damage. Among numerous vasodilators the nitric oxide (NO) family represents an effective means to return blood flow to ischemic tissues (Adams et al., 1997; Homer and Wanstall, 2002; Liu et al., 2016). However, the pharmacological use of NO donors is limited by tolerance or medication side effects (Miller et al., 2000; Lundberg et al., 2009; Mohler et al., 2014). Moreover, endogenous production of NO is attenuated in cardiovascular diseases by reduced expression and uncoupling of its associated enzyme endothelial nitric oxide synthase (eNOS). Alternative methods for increasing endogenous NO (Lohr et al., 2009; Shiva and Gladwin, 2009; Keszler et al., 2018; Wajih et al., 2019; Wajih et al., 2021) have identified nitrite, nitrosyl and S-nitrosothiol (RSNO) containing proteins as potential sources. These compounds do not display pharmacological tolerance, nor do they require optimal intracellular oxygen and pH to function. As an alternative, photo relaxation of conduit vessels in the red or near infrared region of the electromagnetic spectrum has gained growing recognition as a non-invasive targeted modality to increase NO supply (Karu et al., 2005; Poyton and Ball, 2011; Ieda et al., 2020; Colombo et al., 2021).

Photobiomodulation (PMB) or low-level laser (light) therapy (LLLT), is the irradiation of tissues with laser or LED in the visible and near infrared range of the electromagnetic spectrum. The cellular mechanisms of photobiomodulation are well described, e. g. proliferation, migration, cytoprotection, inflammation, which contribute to accelerated tissue repair such as wound healing (Freitas and Hamblin, 2016). Irradiation affects a variety of cellular functions with implication of multiple regulatory mechanisms (Karu, 1999). One potential hypothesis for light’s action attributes a central role of complex IV (cytochrome c oxidase, Cco) of the electron transport chain (Karu, 1999; Wong-Riley et al., 2005; Karu T., 2010; Hamblin, 2016), however, numerous other reaction routes may contribute to the general network. Some pathways involve NO which is canonically produced via the activity of its synthase enzymes but can also enter the cells from outside by diffusion, or from nitrate and nitrite containing nutrients. The latter can be reduced in many ways (Machha and Schechter, 2011), and specifically in the mitochondrion by the reductase activity of Cco (Poyton and Ball, 2011) or the ubiquionone cycle (Kozlov et al., 1999).

670 nm light facilitates the availability of these NO precursors from their stores (Wong-Riley et al., 2005; Machha and Schechter, 2011), and induces NO release outside of the mitochondria related mechanisms from nitrosylated heme (Lohr et al., 2009; Shiva and Gladwin, 2009), non heme dinitrosyl iron complexes (DNIC) or S-nitrosothiols (RSNO) (Keszler et al., 2018; Wajih et al., 2019; Wajih et al., 2021). Red light synergistically enhances the vasodilatory effect of nitrite (Lohr et al., 2009; Wajih et al., 2019; Wajih et al., 2021) and is capable to liberate NO from nitrosyl and nitroso derivatives (Lohr et al., 2009; Keszler et al., 2018). N-nitroso compounds (RNNO) may also be a putative target of photolytic cleavage to NO, however, mainly at shorter wavelengths, and with a minor relaxing effect (Rodriguez et al., 2003).

Red light treatments reduce the damage observed in traumatic brain injury and stroke models (Hamblin, 2016), as well as cardiac ischemia-reperfusion injury through NO mechanisms (Lohr et al., 2009; Shiva and Gladwin, 2009). Small case studies using red light as a therapy for diabetic wounds (Desmet et al., 2006) and chronic venous ulcers (Caetano et al., 2009) suggest clinical relevance.

In earlier studies (Lohr et al., 2009; Keszler et al., 2017; Keszler et al., 2018; Keszler et al., 2019; Weihrauch et al., 2021) we recognized that 670 nm wavelength is optimal to achieve ex vivo dilation of dissected blood vessel and the light effect is not hindered by temperature interference. The vasodilation occurs via activation of the NO pathway. Investigation of the underlying mechanism revealed a transferable species secreted from the endothelium which is stable and active ex vivo for at least for 30 min, and contains NO moiety, and most probably also free NO. The secretion of the dilatory species is under the control of red light.

Here we examined the stability of the endothelium derived NO precursor in an in vivo mouse model by detecting blood flow and measuring the levels of the vasodilatory species in plasma and tissue samples collected at the end point. We optimized the conditions by which 670 nm light would maximize blood flow and determined the stability of the vasoactive NO precursor in plasma and muscle tissue under optimal conditions. Then, in order to mimic the clinical conditions of peripheral artery disease (PAD), we placed an ameroid constrictor, a device containing hygroscopic material to partially occlude the vessel, on the proximal femoral artery of mice. Then measured the arteriolar blood flow reduction and its restoration by red light treatment.

Previously we found that the ex vivo vasodilatory effect of red light (Keszler et al., 2017; Keszler et al., 2019; Weihrauch et al., 2021) is optimal at 670 nm wavelength and occurs via an endothelium derived transferable NO precursor. This precursor is stable and active at least for 30 min under physiological conditions. The long half-life of NO precursors in the bath was explained by their presence in microvesicles which subsequently exit the cell by electromagnetic energy (Weihrauch et al., 2021). Our current study extended the ex vivo investigations to in vivo conditions and convincingly established that 1) the light treatment is optimal at an intensity of 50 mW/cm² for 5–10 min (Figure 1), 2) the NO precursor vasodilator is active and stable for 30 min (Figure 3A), 3) accumulates in the plasma during light exposure, and is quickly drained when the irradiation is finished (Figure 3B). Finally, we mimicked chronic peripheral artery disease by designing and implanting an ameroid constrictor onto the hind limb femoral artery of mice (Figure 5) and restored the restricted blood flow by application of 670 nm light (Figure 6).

Light mediated vasodilation was initially described by Robert Furchgott when UV and blue light induced relaxation of rabbit aorta (Furchgott et al., 1961). Since then, similar studies have shifted to longer wavelengths where the tissue penetration is deeper, and the radiation does not produce harmful side-effects. Most animal studies, ex vivo and in vivo, focus on wound healing and angiogenesis, while only a minor part targets microcirculation or blood pressure (Colombo et al., 2021). Ex vivo light therapy conducted on porcine coronary arteries at 670 and 680 nm wavelengths resulted in a dose dependent relaxation, which was blunted by addition of cPTIO or ODQ suggesting NO-dependent mechanism (Plass et al., 2012). These results are consistent with our previous findings on murine facialis arteries (Keszler et al., 2017). Spontaneously hypertensive rats subjected to in vivo irradiation at 660 nm at six different body points, and 69% of them showed a considerable decrease of systolic arterial pressure, accompanied with an increased total serum NO (nitrite plus nitrate) level (Buzinari et al., 2020). In addition, human applications of PMB research on mitigation of endothelial dysfunction are available both in the far red and near infrared regions (Colombo et al., 2021). Application of 890 nm laser for 30 min on the forearm of volunteer human subjects resulted in an increase of total serum NO content of locally drawn venous blood until 5 min in the treatment followed by a drop during the examined period (Mitchell and Mack, 2013). This pattern is similar to what we found in plasma NO precursor levels when mice received full body exposure of 670 nm LED light.

The mechanism of PMB is complex and highly dependent on the applied wavelength and power (Karu, 1999; Colombo et al., 2021; George et al., 2018). Photo acceptor molecules that absorb specific wavelengths of light participate in regulation of metabolic pathways, therefore the action of light is categorized with primary (direct photochemical) and secondary (downstream) effects. Primary targets including mitochondria, various heme proteins and S-nitrosothiols upon activated trigger signaling pathways leading to alteration of physiological events. The mitochondrion features both categories, since photoactivation of the electron transport chain complexes influences the organelle’s ATP production, calcium flux, or reactive oxygen species levels (Karu T. I., 2010; Amaroli et al., 2021). Noteworthy, that energy in the near infrared region is also absorbed by intracellular water layers (George et al., 2018). Further, photo acceptors outside the mitochondria such as nitrosylated iron complexes and nitroso compounds are also critical transmitters of light mediated physiological events.

According to our earlier proposed mechanism (Weihrauch et al., 2021) 670 nm light induces endothelium derived vesicle formation and subsequent exocytosis. The vesicles are vasoactive and contain NO precursors. Previous observation suggests the most probable vasoactive NO precursor is RSNO (Keszler et al., 2019), however, DNIC is also a plausible candidate (Keszler et al., 2018; Keszler et al., 2019), while RNNO compounds and nitrosylated cytoglobin (Zweier and Ilangovan, 2020) cannot be excluded either. These potential NO precursors were all taken into consideration and quantified with triiodide dependent chemiluminescence without discriminating among them with specific reagents. The NO species unrelated to the endothelium, such as nitrosyl hemoglobin (HbNO) and S-nitroso hemoglobin (Hb-SNO) were not considered relevant in the ex vivo model, hence RBCs were not analyzed. However, participation of species outside the endothelium cannot be excluded from the overall mechanism.

The reported findings support advancing red light as a non-invasive vasodilator, however some experimental limitations in this study remain. The limited plasma volume from mice required exsanguination, therefore locally drawn blood could not be collected to detect sufficient amounts of the NO precursor. Since NO has been implicated as a mediator of red-light stimulated angiogenesis, the relative contribution of angiogenesis to the present observations must be considered. Murine hindlimb angiogenesis is well described 7 days after femoral artery excision and severe ischemia (Lohr et al., 2013). The ameroid model used in this study does not abolish blood flow, therefore the angiogenic stimulus of ischemia is lessened. Therefore, we proffer the acute changes in blood flow at early timepoints (less than 7 days) do not coincide with angiogenesis. The contribution of angiogenesis to blood flow changes at later timepoints are less certain, and we intend to explore the potential role of angiogenesis in ameroid ischemia in the future. Penetration of the light into tissues should be considered if practical therapies are to be developed. The penetration depth of the employed wavelength is reported to be 4–5 mm (Ash et al., 2017) at 10 J/cm², but our Monte Carlo analysis (unpublished data) gave 10% energy retention at 15–20 mm at the applied 22.5 J/cm². This depth range can interact with dermal vasculature. Moreover, this study supports that red light irradiation secretes a vasodilator into the bloodstream. This finding is important when discussing the penetration depth because a humoral factor has the potential to indirectly stimulate vascular function at greater depths. An additional consideration for any therapeutic device design should include an assessment of heat. The LED array was set 1 cm above the target to ensure any heat generation was dissipated by ventilation. Temperature was monitored during the procedure and did not show an increase. Previously we have confirmed that red light illumination does not significantly increase temperature and 37°C temperature only caused less than 1.1% dilation under ex vivo conditions (Keszler et al., 2018). Nevertheless, our overall results support the hypothesis that 670 nm light effectively increases vasodilation by generating a NO-dependent vasodilator in deep tissues, and these findings may serve as a basis for future therapies.

Investigating mechanisms which improve cardiovascular function is vital to developing future therapies for vascular diseases. Low-level red-light therapy increases NO bioavailability locally and most efficiently when enzymatic NO production is compromised during ischemia. PAD is a highly prevalent disease affecting over eight million Americans and generates a high ischemic burden secondary to occlusive arterial disease and endothelial dysfunction (Matsushita et al., 2019). Our murine ameroid constrictor model proved to be a good means to investigate the mitigation of symptoms of this disease. The research presented here focuses on physiological aspects of light delivery and concludes that 670 nm light treatment increases perfusion. Since non-invasive therapies for PAD patients are limited, red-light therapy may offer an effective treatment of individuals with impaired blood flow and tissue ischemia.