Intense light as potential future therapy for myocardial injury in patients after non cardiac surgery: lessons from mice and men

Introduction

Perioperative mortality in patients over 45 years of age is ~1–2% and nearly half of these deaths result from cardiovascular complications (1). In the perioperative period, myocardial injury in noncardiac surgeries (MINS) occurs in approximately 20% of patients (1) and has been shown to significantly increase 1-year mortality rates. A meta-analysis of 37 cohort studies found that the mortality rate for myocardial injury (MI-I) after non-cardiac surgery is 25% at 1 year (2,3). MINS is defined as elevated postoperative cardiac troponin (cTn) due to ischemia, with or without additional symptoms or electrocardiogram (ECG) changes, that occurs during or within 30 days after noncardiac surgery. However, such elevations in cTn nearly always occur within the first 2 postoperative days. Clinical symptoms and electrocardiographic changes are not required to establish a diagnosis of MINS. It has been suggested that endothelial dysfunction is a significant contributor to MINS (4,5). In fact, malfunction of endothelial cells and the associated vasculature are key components in the pathogenesis of a large amount of the most impactful and critical human diseases. Indeed, the endothelium controls essential physiological processes and is considered one of the largest organs, covering 3,000 to 6,000 square meters in surface area in an average-sized person. Defects in the endothelium result in heart disease, peripheral vascular disease, stroke, diabetes, insulin resistance, chronic kidney failure, tumor growth, metastasis, venous thrombosis, and severe viral infectious diseases. Thus, a dysfunctional endothelium is a hallmark of human diseases (6).

In animal studies, circadian enhancement has been associated with improved health, including reversal of a metabolic syndrome (7) and protection from myocardial ischemia (MI) through endothelial barrier protection (8,9). A potent circadian rhythm enhancer is blue and bright light with an intensity of >10,000 lux (lx). In our studies, we have demonstrated that intense blue light enhances circadian rhythms, improves sleep quality, results in favorable metabolic changes, is anti-inflammatory and anti-thrombotic in healthy human volunteers (8,10,11). Our basic science research from the last 2 decades together has revealed that intense light is cardioprotective by improving and boosting the endothelial function (8,12). Through a recent unbiased whole genome array, we discovered the endothelial factor ANGPTL4 to be the top light-responsive protein and to be downstream of our intense light-elicited cardioprotective mechanisms (13). Based on ANGPTL4’s protective function for the endothelium, and the heart (13), we hypothesized that through circadian enhancement with intense light therapy, we could harness the endothelial-, and cardio-protective properties of ANGPTL4 and to use ANGPTL4 as a therapy or biomarker to predict clinical outcomes. In the current studies we addressed several knowledge gaps and limitations of prior studies: Studies on light-elicited cardioprotection identified several downstream targets, however, pharmacological therapies have not been explored yet. Furthermore, intense light in female mice or intense light in a survival model evaluating cardiac function has not been done yet. Although initial translational studies have been conducted with healthy human volunteers, intense light therapy in patients has not yet been performed to evaluate the pathways identified in murine studies. Here we attempted to explore feasibility and to identify biomarkers of light therapy in patients to design a bigger clinical trial on intense light therapy in humans. In the current studies presented, our animal and human studies on intense light exposure are novel, striking, and significant and attempt to close some of the knowledge gaps and limitations of the past. We present this article in accordance with the MDAR reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-25-27/rc).

Methods

In the current manuscript, we used animal studies (in situ MI, survival studies after MI, pharmacological studies during in situ MI, gene target mice during in situ MI), in-vitro experiments (isolated human endothelial cells, isolated mouse aortas) and patient studies (intense light exposure in patients after non cardiac surgery) to evaluate and explore the role of intense light elicited therapies and mechanisms in cardioprotection.

Mouse experiments

Animal experimental protocols (Protocol #1310) were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado Denver, USA, in accordance with the National Institutes of Health (NIH) guidelines for the care and use of live animals. All mice were housed in a 14 h:10 h L(light):D(dark) cycle and we routinely used 12- to 16-week-old male mice. All mice had a C57BL/6J background. C57BL/6J. Per2^loxP/loxP mice were generated by Ozgene (Pert, Australia). VE-Cadherin-Cre [B6.Cg-Tg(Cdh5-cre)7Mlia/J (14)] and C57BL/6J mice were purchased from Jackson laboratories. To obtain endothelial tissue-specific mice, we crossbred Per2^loxP/loxP mice with the VE-Cadherin-Cre recombinase mouse. Before experiments, mice were housed for at least 4 weeks in a 14/10-h light-dark (lights on 6 AM, lights off 8 PM) cycle to synchronize (entrain) the circadian clock of wildtype (WT) mice to the ambient light-dark cycle. We conducted all mouse experiments at the same point [Zeitgeber (ZT)3, ZT15] unless specified otherwise. All animals were under deep anesthesia while undergoing surgical procedures. The depth of anesthesia was regularly controlled. For the induction of anesthesia [intraperitoneal (i.p.), pentobarbital], mice were shortly anesthetized with sevoflurane, ensuring that discomfort, distress, pain, and injury was limited to a minimum.

Intense light exposure

Intense light exposure of mice for seven days (14 h of 10,000 lx and 10 h of 0 lx, Lightbox, Uplift Technologies DL930, full spectrum, UV filter) was compared to room light exposure of mice for seven days [14 h of 200 lx and 10 h of 0 lx]. Previous studies found no temperature changes or effects on cortisol blood levels using intense light exposure up to 7 days in mice (8). Light exposure was conducted in a separate room to avoid disturbance of other animals. The light intensity was measured using a lx meter. During light exposure, mice were housed in their original cages with access to food and water ad libitum.

Murine model for cardiac MI

Murine in situ myocardial ischemia and reperfusion injury (IRI) (60-min ischemia/120 min reperfusion) and troponin-I (cTnI) measurements were performed as described (15-17). We determined infarct sizes by calculating the percentage of infarcted myocardium to the area at risk (AAR) using a double staining technique with Evan’s blue (Sigma-Aldrich, Berlington, USA; Cat# E2129; Cas: 314-13- and triphenyltetrazolium chloride (Sigma-Aldrich, Cat# T8877; Cas: 298-96-4). AAR and the infarct size were determined via planimetry using the NIH software Image 1.0 (National Institutes of Health, Bethesda, MA, USA).

Echocardiography

For echocardiography, mice were anesthetized with 2% isoflurane and cardiac function was assessed by 2D-transthoracic echocardiography using a Visual Sonics Vevo 770 high-resolution ultrasound imager equipped with a 35-MHz transducer. The heart rate was maintained above 500 beats/min throughout (18). Parasternal long axis and multiple short-axis B-mode videos and M-mode images (at the level of the mid-papillary short axis) were routinely acquired. Determinations of ejection fraction (EF) were performed off-line in a blinded mode.

Cell culture

Human microvascular endothelial cells [HMEC-1, American Type Culture Collection (ATCC), ATCC^® CRL-3243] were cultured as described previously (19,20). Prior to all experiments, cells were serum-starved to align circadian rhythms (21).

Transfection with siRNA

We grew HMEC-1 cells to 70% confluency in complete medium with 10% fetal bovine serum (FBS). We then transfected siGENOME SMARTpool siRNA for Per2 (25 nM), siGENOME nontargeting siRNA control (25 nM) into HMEC-1 cells using Dharmafect 1Transfection Reagent (Horizon Discovery, Cambridge, UK) according to the manufacturer’s protocol. 24 h later, we replaced the transfection medium with complete medium that contained 10% FBS. After another 24 h waiting period, we treated the cells with or without 1 µg/mL of Angptl4 for 30 minutes prior to normoxia or hypoxia exposure (22).

Hypoxia exposure

To expose cells to hypoxia, cells were placed in a hypoxia chamber (Coy Laboratory Products Inc., Grass Lake, MI, USA) using preequilibrated hypoxic medium at 1% O₂ for 6 hours.

Transcriptional analysis

We isolated total RNA using the Trizol Reagent (Thermo Fisher Scientific, Waltham, MA, USA; Cat# 15596018), phenol-chloroform extraction, and ethanol precipitation method. We purified the total RNA using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands; Cat# 74106). Finally, we quantified RNA using Nanodrop 2000. Quantification of transcript levels was determined by real-time reverse transcription polymerase chain reaction (RT-PCR) (LightCycler 96; Roche, Rotkreuz, Switzerland). qPCR reactions contained 1× final primer concentration (Thermo Fisher Scientific) with Taqman fast advanced master mix (Thermo Fisher Scientific). Primer sets used were PER2 (Hs01007553_m1), ACTB (4326315E).

Cell permeability—TEER method

HMEC-1 were grown on polycarbonate permeable supports (0.4-µm pore, 6.5-mm diam; Corning Life Sciences, Acton, MA, USA) at a cell density of 1.5×10⁴ cells/well. We then placed confluent HMEC-1 in a hypoxia chamber (Coy Laboratory Products Inc., Grass Lake, MI, USA). In order to have accurate hypoxia exposure time, we replaced the medium with preequilibrated hypoxic medium at 1% O₂. Finally, we measure the transendothelial electrical resistance (TEER) using the EVOM2 voltohmmeter (World Precision Instrument, Sarasota, FL, USA), subtracting the value of blank wells.

Light therapy in patients undergoing non-cardiac surgery

Patients were exposed to intense light (10,000 lx) every morning for 5 days for a total duration of 30 minutes at sunrise. We collected daily 5 mL of blood before and after light exposure. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Colorado Multiple Institutional Review Board (COMIRB #13-1607), and informed consent was obtained from all individual participants. A total of 15 patients undergoing spine surgery, 8 treated and 7 controls, were enrolled. Control group: average age 69.1 years old, 5 females, 2 males; treated group: average age 67.1 years old, 5 males, 3 females (see Table 1 for patient characteristics). The protocol of the human studies is registered at https://clinicaltrials.gov/study/NCT03822949.

Vasoreactivity of aortas ex vivo using intact and denuded vessel myography

Aortic rings from control animals were pre-contracted with phenylephrine in the presence or absence of ANGPTL4 and relaxed with 10 µM acetylcholine (Ach) to quantify endothelial function. Briefly, thoracic aortae were removed from rodents and placed in an oxygenated Krebs-Heinsleit (KH) buffer. Rings (2 mm) were cut, and in one set of rings, the endothelium was removed by gently rubbing (23,24). Rings were hung on a myograph using triangular hooks and a resting tension of 1 g was applied. After equilibration at 37 °C in KH, aortic rings were stimulated with high potassium KH to assess viability. Rings were relaxed and then contracted with phenylephrine (40 nM). After relaxation, rings were pre-incubated with ANGPTL4 (1 and 10 ng/mL) for 30 min before contraction with phenylephrine and subsequent addition of acetylcholine to assess relaxation (see schematic). To assess chrono release of ANGPTL4 on vasoreactivity, aortae from the control rodents above were harvested and assessed for in vitro responsiveness to acetylcholine in intact and denuded arteries.

Human ANGPTL4 and troponin plasma levels

ANGPTL4 and troponin levels were measured using enzyme-linked immunosorbent assay (ELISA) per manufacturer’s instructions. For ANGPTL4 the RayBio^® Human ANGPTL4 ELISA Kit for cell culture supernatants, plasma, and serum samples was used (Ray Biotech, Peachtree Corners, USA; Cat# ELH-ANGPTL4-1). For cTn levels, we used the cTnI ELISA kit: Human Cardiac Troponin I ELISA Kit from MyBioSource (MyBioSource, San Diego, USA; Cat# MBS729403). Melatonin levels were quantified using MyBioSource Human MT (Melatonin) ELISA Kit (MyBioSource; Cat# MBS704506).

Statistical analysis

For data analyses of two groups, we used the unpaired student, two-sided t-test. If we compared multiple groups, we used a one-way analysis of variance with a Tukey’s post hoc test. We showed all values as mean ± SD and we considered a P<0.05 as statistically significant. We determined group size using BiAS for Windows utilizing effect sizes and standard deviations from preliminary data sets. An a priori sample size analysis for infarct sizes was based on previous studies suggesting a standard deviation for infarct size of 5%. A biologically relevant difference of at least 20% for infarct size between control and experimental groups resulted in 3 animals obtaining statistically significant results with a probability of 0.9 (Figures 1,2). An a priori sample size analysis for TEER was based on previous studies suggesting a standard deviation for TERR of 6 ohms/cm². A biologically relevant difference of at least 30 ohms/cm² for infarct size between control and experimental groups resulted in 3 individual samples per group obtaining statistically significant results with a probability of 0.9 (Figure 3). An a priori sample size analysis for endothelial relaxation in % was based on previous studies suggesting a standard deviation of 11%. A biologically relevant difference of at least 20% for endothelial relaxation between control and experimental groups resulted in 9 individual samples per group obtaining statistically significant results with a probability of 0.9 (Figure 4). An a priori sample size analysis for EF in % was based on previous studies suggesting a standard deviation of 6%. A biological relevant difference of at least 20% for infarct size between control and experimental groups resulted in 3 animals obtaining statistically significant results with a probability of 0.9 (Figure 5A). A biological relevant difference of at least 25% for EF between control and experimental groups resulted in 3 individual animals per group obtaining statistically significant results with a probability of 0.9 (Figure 5B). In the current study no ‘missing data’ occurred. No unexpected or adverse events were observed. All experiments were blindly randomized to experimental or control groups. All animal experiments were terminal experiments. All data describe biological replicates. Replicates are indicated with each figure. Human data are results from a pilot study and as such were not appropriately powered. All data analysis was performed by an independent investigator blinded to the experimental protocol. Data are expressed as mean ± SD. For statistical analysis GraphPad Prism 5.0 software for Windows or Bias for Windows^® (epsilon-Verlag, Frankfurt, Germany) was used. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

Figure 1 The circadian PER2 amplitude enhancer nobiletin and the HIF1A stabilizer DMOG during MI in Per2 endothelial specific deficient mice. (A) Per2^loxP/loxP or Per2^loxP/loxP-VE-Cadherin-Cre mice were treated i.a. with vehicle [solutol in 0.9% NaCl (ratio 1:100)] or nobiletin in solutol:0.9% NaCl (ratio 1:100; 1 mg/kg) at the onset of reperfusion following MI. MI consisted of 1 h of ischemia followed by 2 h of reperfusion. Representative infarct sizes measured by double staining with Evan’s blue and TTC are shown. Infarct sizes are expressed as the percentage of the AAR that underwent infarction. (B) Representative infarct sizes. (C) Per2^loxP/loxP or Per2^loxP/loxP-VE-Cadherin-Cre mice were treated i.a. [intraarterial (into the carotid artery)] with vehicle (NaCl) or DMOG [1 mg per mouse (25)] at the onset of reperfusion following MI. MI consisted of 1 h of ischemia followed by 2 h of reperfusion. Infarct sizes were measured by double staining with Evan’s blue and TTC and are expressed as the percentage of the AAR that underwent infarction. (D) Representative infarct sizes. V, vehicle; N, nobiletin; D, DMOG; data are presented as mean ± standard deviation; n=7 individual mice per control group, n=3 individual mice per treatment group; all mice were male littermates. ns, not significant; **, P<0.01; ***, P<0.001; ****, P<0.0001. AAR, area at risk; DMOG, dimethyloxalylglycine; HIF1A, hypoxia inducible factor 1 alpha; i.a., intraarterial; MI, myocardial ischemia; NaCl, sodium chloride; PER2, period circadian regulator 2; TTC, 2,3,5-triphenyltetrazolium chloride.

Figure 2 ANGPTL4 restores a wildtype phenotype in endothelial Per2 tissue specific mice. (A) Per2^loxP/loxP or Per2^loxP/loxP-VE-Cadherin-Cre mice were treated intraarterial with vehicle (NaCl) or murine ANGPTL4 (10 µg/kg) at the onset of 2 h reperfusion following 1 h of myocardial ischemia. Infarct sizes are expressed as the percent of the area at risk that underwent infarction. (B) Representative infarct sizes. Data are presented as mean ± standard deviation; n=5 individual mice per control group, n=3 individual mice per treatment group; all mice were male littermates. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. NaCl, sodium chloride.

Figure 3 ANGPTL4 restores a wildtype phenotype in hypoxia exposed human PER2KD endothelial cells. Permeability assay in PER2KD or Scr HMEC-1 during 24 h of 1% hypoxia treated with vehicle (A) or recombinant ANGPTL4 protein (B) (data are presented as mean ± standard deviation; n=3 individual experiments for control and experimental group). Note that permeability increases after prolonged hypoxia exposure of endothelial cells due to morphological changes. KD, knockdown; PER2, period circadian regulator 2; TEER, transepithelial electrical resistance.

Figure 4 Endothelial-dependent vasodilation is potentiated by ANGPTL4. Aortic rings from control animals were pre-contracted with phenylephrine in the presence or absence of ANGPTL4 and relaxed with 10 µM Ach. (A,B) ANGPTL4 exposure results in a dose-dependent potentiation of Ach-mediated relaxation. Acute treatment (A) did not result in significant changes, however, 24 h pretreatment with ANGPTL4 (B) significantly increased acetylcholine mediated relaxation. Data are presented as mean ± standard deviation; n=21 individual isolated aortic rings for the control group, n=10 individual isolated aortic rings for the 1 ng/mL ANGPTL4 treated group and n=17 individual isolated aortic rings in the 10 ng/mL ANGPTL4 treated group. ns, not significant; **, P<0.01. Ach, acetylcholine.

Figure 5 No sex difference in intense light elicited cardioprotection and post-MI intense light treatment improves cardiac function in wild type mice. (A) MI consisted of 1 h of ischemia followed by 2 h of reperfusion in female wildtype mice. Representative infarct sizes measured by double staining with Evan’s blue and TTC are shown. Infarct sizes are expressed as the percent of the AAR that underwent infarction; data are presented as mean ± standard deviation; n=5 individual mice per control group [room light], n=4 individual mice per treatment group; all mice were female littermates. (B) EF % in C57BL/6J at baseline (C, control/sham) or after 45 min MI followed by 1-week reperfusion under room (RL) or intense light (IL) housing conditions. LD 14:10 h, data are presented as mean ± standard deviation; n=4 individual mice per control group, n=3 individual mice per treatment group; all mice were male littermates. ns, not significant; *, P<0.05; **, P<0.01. AAR, area at risk; EF, ejection fraction; LD, light:dark; MI, myocardial ischemia; TTC, 2,3,5-triphenyltetrazolium chloride.

Results

Cardioprotection via the circadian amplitude enhancer nobiletin (NOB) requires endothelial expressed PER2

Based on studies showing that intense light increased the circadian amplitude via PER2, and that intense light mediated cardioprotection is endothelial PER2 specific, we evaluated pharmacological approaches to mimic light elicited PER2 in cardioprotection. Recent studies identified several circadian amplitude-enhancing small molecules in a high throughput screen (26). One of the natural compounds identified was the flavonoid NOB, which robustly enhanced the amplitude of PER2 (7,27).

Since we had previously demonstrated that intense light-elicited cardioprotection requires endothelial PER2, NOB mediated circadian amplitude enhancement and cardioprotection (28) would thus be expected to require endothelial expressed PER2. To evaluate a potential NOB-specificity, we treated control (Per2^loxP/loxP) or endothelial specific PER2 knockout (Per2^loxP/loxP-VE-Cadherin-Cre) mice with NOB following 60 minutes of MI. As shown in Figure 1A,1B, NOB significantly reduced infarct sizes in control mice. However, NOB mediated cardioprotection was entirely abolished in Per2^loxP/loxP-VE-Cadherin-Cre mice. Together, these data demonstrate that the PER2 amplitude enhancer NOB requires endothelial expressed PER2 to mediate cardioprotection.

Cardioprotection via the hypoxia inducible factor 1 alpha (HIF1A) stabilizer DMOG requires endothelial PER2

HIF1A and the circadian system are interconnected as HIF1A and the circadian core regulators are capable of direct binding to each other (29). While PER2 regulates the HIF1A response, HIF1A also controls the circadian machinery (30). As such hypoxia changes the core clock gene expression in a time- and tissue-dependent manner (31). How individual drugs, targeting one pathway, may lead to changes in the other pathway are unknown (30). While we found hypoxia-induced HIF1A transcription of the oxygen-efficient mitochondrial gene Cox4.2 to be abolished in a human microvascular endothelial PER2 knockdown cell line (8), administration of the HIF1A stabilizer dimethyloxalylglycine (DMOG) has never been investigated in Per2 deficiency. Accordingly, we treated Per2^loxP/loxP or Per2^loxP/loxP-VE-Cadherin-Cre mice with DMOG following 60 minutes of MI. As shown in Figure 1C,1D, we were able to demonstrate that DMOG-mediated cardioprotection was abolished in Per2^loxP/loxP-VE-Cadherin-Cre mice. Together, these data demonstrate that DMOG-mediated cardioprotection requires endothelial-expressed PER2.

ANGPTL4 as a downstream target of PER2-HIF1A in cardioprotection

To understand intense light-elicited and PER2-dependent pathways, we recently conducted a genome-wide array, analyzing intense light-dependent gene expression before any ischemia even occurred. In silico analyses found dominant regulation of circadian pathways and identified the HIF1A-regulated angiopoietin-like 4 (ANGPTL4) as the top light and PER2-dependent gene. As ANGPTL4 was found to be downstream of PER2/HIF1A we hypothesized that ANGPTL4 treatment could overcome a Per2 deficiency in mice. In proof-of-concept studies, we treated Per2^loxP/loxP or Per2^loxP/loxP-VE-Cadherin-Cre with recombinant ANGPTL4 (10 microg/kg) during reperfusion. Indeed, as shown in Figure 2, recombinant ANGPTL4 restored a wild-type phenotype in Per2^loxP/loxP-VE-Cadherin-Cre and provided robust cardioprotection in control mice. Together, these data demonstrate that ANGPTL4 mediates cardioprotection in wildtype and endothelial-specific-Per2-deficient mice.

ANGPTL4 restores a PER2 knockdown mediated increase of cell permeability in a human cell line

Based on our findings for a critical role of light-elicited PER2 for endothelial barrier protection in vivo, we next evaluated the role of ANPTL4 on cell permeability in vitro using a PER2 knockdown (KD) HMEC line (HMEC-1) (32). As previous studies found that ANGPTL4 provides endothelial barrier protection and is light and PER2 dependent, we exposed control or PER2KD HMECs to hypoxia. Hypoxia results in a barrier disruption as seen during myocardial IRI. Using a trans-endothelial electrical resistance (TEER) permeability assay (8), we found significantly increased cell permeability in hypoxic PER2KD vs. control HMEC-1 (Figure 3A). We next treated our cells with recombinant ANGPTL4 treatment (1 microg/mL) and again exposed control or PER2KD HMECS to hypoxia. As shown in Figure 3B, ANGPTL4 treatment was able to restore a wildtype phenotype in PER2KD HMECS. Together, these data demonstrate that ANGPTL4 can overcome a PER2 deficiency in human endothelial cells and protect the endothelial barrier from hypoxia-elicited barrier disruption.

ANGPTL4 pretreatment increases acetylcholine-mediated vessel relaxation

Endothelial function has been shown to be impaired in patients after MI (33) and is associated with a reduction in circulating nitric oxide (34). To further investigate the effects of ANGPTL4 on endothelial function, we next measured vasoreactivity of aortas ex vivo using intact and denuded vessel myography (35). As shown in Figure 4A, ANGPTL4 potentiated acetylcholine-induced relaxation in a dose-dependent manner, suggesting an endothelial-dependent mechanism. However, these effects were not statistically significant. As such, we tested the hypothesis that, like light pretreatment, an ANGPTL4 pretreatment would be necessary in order to observe significant changes. Indeed, 10 ng/mL ANGPTL4 pretreatment of aortas ex vivo resulted in a significant increase of acetylcholine-induced relaxation when compared to vehicle-treated controls (Figure 4B). Furthermore, removal of the endothelium completely abolished the responsiveness to both acetylcholine and ANGPTL4, suggesting an endothelial-dependent mechanism (data not shown). Together, these data demonstrate that ANGPTL4 improves vessel relaxation ex vivo.

Intense light exposure before or after heart ischemia is cardioprotective

Considering that housing male mice under intense light conditions increased the circadian amplitude and decreased infarct sizes in previous studies, we hypothesized that we would find similar effects of light therapy in female mice. In fact, as shown in Figure 5A,5B, following 7 days of 14 h light followed by 10 h darkness [light:dark (LD) cycle 14:10 h], infarct sizes were significantly decreased in female mice housed under intense light conditions when compared to female mice housed under standard room light conditions.

Next, we were curious if intense light therapy could also be beneficial following MI. Indeed, post-MI intense light treatment has never been investigated. Thus, we used a survival model for MI and repression injury in mice and determined the EF using echocardiography 5 days post-MI. A shown Figure 5C, our data show significantly improved myocardial function—as shown by the echocardiography Vevo770 (VisualSonics) system—in C57BL/6J mice that received intense light during one week of reperfusion following 45 minutes of MI. Based on these studies, we concluded that intense light protects female mice like male mice and that intense light therapy after MI improves the EF. Overall, intense pre- and post-treatment provides robust cardio protection in mice.

Intense light therapy increases ANGPTL4 and decreases troponin in patients undergoing non-cardiac surgery

Following our murine studies, we were curious if intense light could promote cardio protection in the perioperative setting. Troponin levels on postoperative day 1 through 3 are indicative of MI-I (MINS) and are predictive of mortality (36). Recently, we evaluated the incidence of MINS in the perioperative setting. The data were obtained from the Multicenter Perioperative Outcomes Group (MPOG) and included data from 1,773,118 patients across 50 institutions between 2014 to 2019. Here we were able to demonstrated that MINS has as a circadian pattern (37). As such, we hypothesized that intense light therapy-mediated circadian amplitude enhancement would reduce MINS in patients undergoing non-cardiac surgery (see patient characteristics in Table 1). We performed intense light therapy starting on postoperative day 1. We collected blood before and after light therapy and analyzed plasma samples. Here, similarly to what we had seen in mice, all patients who were exposed to intense light showed a significant increase of ANGPTL4 plasma levels (Figure 6A).

Figure 6 Intense light therapy after non cardiac surgery increases ANGPTL4 and melatonin but decreases troponin levels in plasma samples. ANGPTL4 (A), melatonin (B) and troponin (C) plasma levels in high-risk patients after spine surgery with and without intense blue light therapy. Treated patients were exposed to 30 min of intense blue light (10,000 lx) in the morning on 5 consecutive days. Blood drawings were performed before and immediately after light exposure. n=15 patients with ICU admission after spine surgery, 8 treated, 7 controls, control group: average age 69.1 years old, 5 females, 2 males; treated group: average age 67.1 years old, 5 males, 3 females. **, P<0.01; ****, P<0.0001. ICU, intensive care unit.

Next, we analyzed melatonin levels, a marker of intense light-regulated circadian rhythms. In fact, previous studies have shown that intense light suppresses melatonin in healthy volunteers (8). Surprisingly, we found the opposite kinetics in patients undergoing non-cardiac surgery. Intense light significantly increased melatonin levels, as shown in Figure 6B.

Finally, we analyzed troponin levels as marker of MINS. Using intense light therapy in the postoperative period in patients undergoing complex spine surgery, we found that patients treated with intense light demonstrated a decrease in their serum troponin levels of nearly 20% through post-operative day 2, whereas untreated patients demonstrated a 200% increase in serum troponin (Figure 6C). These data strongly suggest that intense light therapy may represent a possible novel strategy to reduce perioperative MINS and associated mortality. Together, we found that intense light therapy in patients undergoing non cardiac surgery increases ANGPTL4 and Melatonin plasma levels but decreases troponin levels.

Discussion

In animal studies, circadian enhancement has been associated with improved health, including reversal of a metabolic syndrome (7) and protection from MI through endothelial barrier protection (8). A potent circadian rhythm enhancer is blue and bright light with an intensity of >10,000 lx. In our previous studies, we have demonstrated that intense blue light enhances circadian rhythms, improves sleep quality, results in favorable metabolic changes, is anti-inflammatory and anti-thrombotic in healthy human volunteers (8,10,11).

Our basic science research from the last two decades together has revealed that intense light works through improving and boosting the endothelial function (8). Through a recent unbiased whole genome array, we discovered the endothelial factor ANGPTL4 to be the top light-responsive protein and to be downstream of our intense light elicited protective mechanisms (13).

In the current manuscript, we evaluated further light-elicited mechanisms in mice and men. We were able to show that ANGPTL4 is indeed a downstream target of intense light elicited PER2/HIF1A and can overcome and treat a PER2 deficiency. Furthermore, our research data demonstrates that intense light therapy is associated with a significant increase of ANGPTL4 in animals and in patients. As such ANGPLT4 might be a promising marker of effective light therapy in patients. Moreover, intense light therapy was found to protect mice following MI or was able to reduce troponin levels in the postoperative period in patients undergoing spine surgery. These data strongly suggest that intense light could reduce the incidence of MINS.

The relationship between HIF1A and PER2 is well established (21,38-43). HIF1A’s cardioprotective role is well documented (21,25,44-54), and HIF1A activators have been advocated as therapy for myocardial IRI and cardiac repair (55). Our research found that light increases the amplitude of HIF1A-regulated pathways. However, no studies have been undertaken to study the role of drug-induced HIF1A to mediate cardioprotection in the setting of Per2 deficiency. HIF1A and the circadian system are interconnected as HIF1A and circadian core regulators are capable of direct binding to each other (29). While PER2 regulates the HIF1A response, HIF1A also controls the circadian machinery (30). In fact, hypoxia changes the gene expression of clock genes in a time- and tissue-dependent manner (31). How individual drugs (DMOG/NOB), targeting one pathway, may lead to changes in the other pathway is unknown (30). While we found hypoxia-induced HIF1A transcription of the oxygen efficient mitochondrial gene Cox4.2 to be abolished in human endothelial PER2KD (8), DMOG administration as a strategy to overcome a Per2 deficiency has never been investigated. In fact, we were able to demonstrate that a single DMOG dose during reperfusion following MI can reduce infarct sizes in control mice but not in Per2^loxP/loxP-VE-Cadherin-Cre mice. These data demonstrate that the HIF1A stabilizer DMOG requires endothelial expressed PER2 and is not able to overcome an endothelial Per2 deficiency. These data are in support of previous findings where HIF1A transcription of important cell protective genes was found to be abolished.

Virtually nothing is known about the mechanisms of light-elicited circadian amplitude enhancement and cardioprotection. Using a genome-wide screen, we identified the barrier-protective, endothelial-expressed, and HIF1A-regulated ANGPTL4 as light and PER2-dependent protein. While circadian amplitude-enhancing strategies are an area of intense investigation (56), nothing is known about ANGPTL4 as an underlying mechanism. However, while ANGPTL4 administration has been shown to improve the endothelial barrier (57,58), no study has evaluated ANGPTL4 as downstream target of PER2 in myocardial IRI (59). In proof-of-concept studies, we therefore treated Per2^loxP/loxP-VE-Cadherin-Cre mice with recombinant ANGPTL4 during reperfusion and determined infarct sizes. These data demonstrated that recombinant ANGPTL4 restored a wildtype phenotype in Per2^loxP/loxP-VE-Cadherin-Cre and provided robust cardioprotection in controls. Together, these data highly suggest that ANGPLT4 is indeed downstream of PER2 and has the potential to restore circadian Per2 deficiency.

Previous studies on light-elicited cardioprotection were all done in male mice and published data indicate sex differences in PER2 (60) or HIF1A (61) pathways. As such, we also investigated intense light therapy in female mice. Here, we were able to confirm that housing female mice for 1 week under intense light conditions was similarly cardioprotective as observed in male mice. This finding suggest that intense light mediated cardioprotection is mostly independent of female hormonal pathways.

As light pretreatment might not be feasible in a clinical setting, we also investigated if intense light exposure following MI could be potentially protective. We chose to use echocardiography as a readout as it is most clinically relevant. Indeed, housing our mice under intense light conditions was able to improve EFs almost similar to controls without ischemia. Based on these data, we were further encouraged to utilize intense light in a clinical setting.

In our small pilot study using intense light therapy in high-risk patients undergoing complex spine surgery, we performed intense light therapy (10,000 lx; blue light) starting on postoperative day 1. We collected blood before and after light therapy and analyzed these plasma samples. We learned from these studies that we can use the endothelial marker ANGPTL4 as a biomarker of intense light therapy. Similarly, to what we’ve seen in mice, all patients who were exposed to intense light showed increased ANGPTL4 plasma levels. We further learned that melatonin, a marker of intense light-regulated circadian rhythms, might have the same kinetics. These data were quite surprising as intense light suppresses melatonin in healthy volunteers (8), which, however, does not seem to be the case in patients undergoing non-cardiac surgery. Interestingly, these data may explain why so many intense light studies have not been effective in the past, as many previous studies used melatonin as a biomarker, which does not follow the expected kinetic. Indeed, it has been suggested that critically ill patients have a dampened, or even lost, circadian rhythm due to significant physiologic stress and that increasing melatonin may be beneficial for restoration of normal circadian rhythms (62), which could also be the case in patients after surgery. Lastly, troponin levels on postoperative day 1 through 3 are indicative of MINS and are predictive of mortality (36). However, there is no therapy for MINS available.

Recently we evaluated the incidence of MINS in the perioperative setting. The data were obtained from the MPOG and included data from 1,773,118 patients across 50 institutions between 2014 to 2019. Here we were able to demonstrate that MINS has as a circadian pattern (37). In our pilot study using intense light therapy in high-risk patients undergoing complex spine surgery, we found that patients treated with intense light demonstrated a decrease in their serum troponin levels of nearly 20% through post-op day 2, whereas untreated patients demonstrated a 200% increase in serum troponin. Together, these data begin to mechanistically define the role of key regulators of circadian signaling and strongly suggest that intense light therapy can be used to prevent perioperative MINS—a disease without therapy.

Limitations

Our animal studies have several limitations, as most myocardial IRI studies were not done using a survival model. However, we have used intense light therapy in a survival model and have shown that intense light can improve cardiac function after heart ischemia. The animal experiments primarily used male mice, with limited data on female mice, potentially limiting the generalizability of the results to both sexes. However, we provide significant data that intense light is cardioprotective in female mice, which suggests that similar mechanisms could be found in both sexes.

Nevertheless, how ANGPTL4 or endothelial function can play a role in improving cardiac function after MI still needs to be explored. Further limitations are that our human studies were not powered to establish the efficacy of intense light therapy. However, the intent of the human study was not to establish intense light as therapy but to determine feasibility of intense light therapy in a clinical setting. As such, seeing significant data for ANGPLT4 being regulated by intense light is striking. The trend for troponin levels is interesting but would need a clinical trial for further validation.

Finally, the current study did not explore the long-term effects of intense light therapy or the specific mechanisms by which ANGPTL4 improves endothelial function or cardiac outcomes. Future studies will have to dedicate significant time and effort to understanding the long-term effects of light therapy.

Conclusions

In summary, based on our animal studies, intense light or intense light-elicited therapies (ANGPTL4 protein) could be promising strategies to reduce MI-I in patients: we have started using intense light therapy in patients and have seen that similar pathways are activated in patients as seen in animals. A clinical trial will be necessary to understand the real impact of intense light therapy on MINS in patients undergoing non-cardiac surgery. Moreover, further research with larger, more diverse human cohorts and long-term follow-up is needed to validate these findings and develop targeted therapies.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-25-27/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-27/dss

Peer Review File: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-27/prf

Funding: This study was supported by the National Heart, Lung, and Blood Institute (NIH-NHLBI) (R56HL156955 to T.d.l.G.E.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-25-27/coif). T.d.l.G.E. serves as an unpaid editorial board member of Annals of Translational Medicine from November 2023 to October 2025. T.d.l.G.E. reports receiving funding from the National Heart, Lung, and Blood Institute under Award Number: R56HL156955 for this study. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Colorado Multiple Institutional Review Board (COMIRB #13-1607), and informed consent was obtained from all individual participants. Animal experimental protocols (Protocol #1310) were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado Denver, USA, in accordance with the National Institutes of Health (NIH) guidelines for the care and use of live animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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