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.

The HFSA Advanced Heart Failure and Transplant Cardiology Fellowship Consensus Conference.

There is waning interest among cardiology trainees in pursuing an Advanced Heart Failure/Transplant Cardiology (AHFTC) fellowship as evidenced by fewer applicants in the National Resident Matching Program match to this specialty. This trend has generated considerable attention across the heart failure community. In response, the Heart Failure Society of America convened the AHFTC Fellowship Task Force with a charge to develop strategies to increase the value proposition of an AHFTC fellowship. Subsequently, the HFSA sponsored the AHFTC Fellowship Consensus Conference April 26-27, 2023. Before the conference, interviews of 44 expert stakeholders diverse across geography, site of practice (traditional academic medical center or other centers), specialty/area of expertise, sex, and stage of career were conducted virtually. Based on these interviews, potential solutions to address the declining interest in AHFTC fellowship were categorized into five themes: (1) alternative training pathways, (2) regulatory and compensation, (3) educational improvements, (4) exposure and marketing for pipeline development, and (5) quality of life and mental health. These themes provided structure to the deliberations of the AHFTC Fellowship Consensus Conference. The recommendations from the Consensus Conference were subsequently presented to the HFSA Board of Directors to inform strategic plans and interventions. The HFSA Board of Directors later reviewed and approved submission of this document. The purpose of this communication is to provide the HF community with an update summarizing the processes used and concepts that emerged from the work of the HFSA AHFTC Fellowship Task Force and Consensus Conference.

Left atrial appendage structural characteristics predict thrombus formation.

INTRODUCTION: Nonvalvular atrial fibrillation (NVAF) is a highly prevalent arrhythmia where loss of synchronized atrial contraction increases the risk of intracardiac thrombus particularly within the left atrial appendage (LAA). Anticoagulation is the mainstay of stroke prevention based on the CHA2 DS2 -VASc score; however, it does not account for LAA structural characteristics. METHODS: The research comprises a retrospective matched case-control study of 196 subjects with NVAF who underwent transesophageal echo (TEE). The control group, without thrombus (n = 117), was selected from two different groups, both pools had: NVAF and CHA2 DS2 -VASc score ≥ 3. One group underwent screening TEE before Watchman closure device placement from January 2015 to December 2019 (n = 74) the second underwent TEE before cardioversion from February to October 2014 (n = 43). The study group, with thrombus (n = 79), included patients with NVAF, TEE study performed between February 2014 and December 2020, and LAA thrombus. The propensity score method was utilized to determine the matched controls while accounting for confounding from prognostic variables resulting in 61 matched pairs included in the analysis data set. LAA ostial area (OA) (calculated from orthogonal measurements 0°, 90° or 45°, 135°), LAA maximal depth, and peak LAA outflow velocity were measured. RESULTS: Patient characteristics and TEE data were collected and compared using the t test or χ2 analysis. We observed a lower LAA peak exit velocity in the thrombus group as compared to the control group. Additionally, we found that patients in the thrombus group had smaller LAA OA at 0° and 90°, at 45° and 135°, using largest diameter, as well as using aggregate OA, and smaller maximum LAA depth compared to patients in the control group. Candidate conditional logistic regression models for the outcome of the presence of thrombus were evaluated. Statistical results from the best-fitting conditional regression model were calculated showing a significant association between aggregate OA and LAA exit velocity with presence of thrombus. CONCLUSION: Utilizing LAA structural characteristics to predict thrombus formation may help refine current cardioembolic stroke (CES) risk estimation.

Successful infusional 5-fluorouracil administration in a patient with vasospastic angina.

A 48-year-old female with metastatic colon adenocarcinoma and history of pre-existing coronary vasospasm with ventricular tachycardia (VT) successfully tolerated de novo 5-fluorouracil (5-FU) chemotherapy infusions with prophylactic administration and optimization of anti-spasm medications. 5-FU has been reported to produce severe cardiotoxic side effects, including coronary vasospasm, ventricular arrhythmias, and sudden cardiac death, and is not typically reported in individuals with pre-existing coronary vasospasm.

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.

Lisinopril Mitigates Radiation-Induced Mitochondrial Defects in Rat Heart and Blood Cells.

The genetic bases and disparate responses to radiotherapy are poorly understood, especially for cardiotoxicity resulting from treatment of thoracic tumors. Preclinical animal models such as the Dahl salt-sensitive (SS) rat can serve as a surrogate model for salt-sensitive low renin hypertension, common to African Americans, where aldosterone contributes to hypertension-related alterations of peripheral vascular and renal vascular function. Brown Norway (BN) rats, in comparison, are a normotensive control group, while consomic SSBN6 with substitution of rat chromosome 6 (homologous to human chromosome 14) on an SS background manifests cardioprotection and mitochondrial preservation to SS rats after injury. In this study, 2 groups from each of the 3 rat strains had their hearts irradiated (8 Gy X 5 fractions). One irradiated group was treated with the ACE-inhibitor lisinopril, and a separate group in each strain served as nonirradiated controls. Radiation reduced cardiac end diastolic volume by 9-11% and increased thickness of the interventricular septum (11-16%) and left ventricular posterior wall (14-15%) in all 3 strains (5-10 rats/group) after 120 days. Lisinopril mitigated the increase in posterior wall thickness. Mitochondrial function was measured by the Seahorse Cell Mitochondrial Stress test in peripheral blood mononuclear cells (PBMC) at 90 days. Radiation did not alter mitochondrial respiration in PBMC from BN or SSBN6. However, maximal mitochondrial respiration and spare capacity were reduced by radiation in PBMC from SS rats (p=0.016 and 0.002 respectively, 9-10 rats/group) and this effect was mitigated by lisinopril (p=0.04 and 0.023 respectively, 9-10 rats/group). Taken together, these results indicate injury to the heart by radiation in all 3 strains of rats, although the SS rats had greater susceptibility for mitochondrial dysfunction. Lisinopril mitigated injury independent of genetic background.

Electromagnetic energy (670 nm) stimulates vasodilation through activation of the large conductance potassium channel (BKCa).

INTRODUCTION: Peripheral artery disease (PAD) is a highly morbid condition in which impaired blood flow to the limbs leads to pain and tissue loss. Previously we identified 670 nm electromagnetic energy (R/NIR) to increase nitric oxide levels in cells and tissue. NO elicits relaxation of smooth muscle (SMC) by stimulating potassium efflux and membrane hyperpolarization. The actions of energy on ion channel activity have yet to be explored. Here we hypothesized R/NIR stimulates vasodilation through activation of potassium channels in SMC. METHODS: Femoral arteries or facial arteries from C57Bl/6 and Slo1-/- mice were isolated, pressurized to 60 mmHg, pre-constricted with U46619, and irradiated twice with energy R/NIR (10 mW/cm2 for 5 min) with a 10 min dark period between irradiations. Single-channel K+ currents were recorded at room temperature from cell-attached and excised inside-out membrane patches of freshly isolated mouse femoral arterial muscle cells using the patch-clamp technique. RESULTS: R/NIR stimulated vasodilation requires functional activation of the large conductance potassium channels. There is a voltage dependent outward current in SMC with light stimulation, which is due to increases in the open state probability of channel opening. R/NIR modulation of channel opening is eliminated pharmacologically (paxilline) and genetically (BKca α subunit knockout). There is no direct action of light to modulate channel activity as excised patches did not increase the open state probability of channel opening. CONCLUSION: R/NIR vasodilation requires indirect activation of the BKca channel.

Red light stimulates vasodilation through extracellular vesicle trafficking.

Red light (670 nm) promotes ex vivo dilation of blood vessels in a nitric oxide (NO) dependent, but eNOS independent manner by secreting a quasi-stable and transferable vasoactive substance with the characteristics of S-nitrosothiols (RSNO) from the endothelium. In the present work we establish that 670 nm light mediated vasodilation occurs in vivo and is physiologically stable. Light exposure depletes intracellular S-nitroso protein while concomitantly increasing extracellular RNSO, suggesting vesicular pathways are involved. Furthermore, we demonstrate this RSNO vasodilator is embedded in extracellular vesicles (EV). The action of red light on vesicular trafficking appears to increase expression of endosome associated membrane protein CD63 in bovine aortic endothelial cells, enhance endosome localization in the endothelium, and induce exit of RSNO containing EVs from murine facialis arteries. We suggest a mechanism by which the concerted actions of 670 nm light initiate formation of RSNO containing EVs which exit the endothelium and trigger relaxation of smooth muscle cells.

Missed opportunities to prevent cardiovascular disease in women with prior preeclampsia.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death in women in every major developed country and in most emerging nations. Complications of pregnancy, including preeclampsia, indicate a subsequent increase in cardiovascular risk. There may be a primary care provider knowledge gap regarding preeclampsia as a risk factor for CVD. The objective of our study is to determine how often internists at an academic institution inquire about a history of preeclampsia, as compared to a history of smoking, hypertension and diabetes, when assessing CVD risk factors at well-woman visits. Additional aims were (1) to educate internal medicine primary care providers on the significance of preeclampsia as a risk factor for CVD disease and (2) to assess the impact of education interventions on obstetric history documentation and screening for CVD in women with prior preeclampsia. METHODS: A retrospective chart review was performed to identify women ages 18-48 with at least one prior obstetric delivery. We evaluated the frequency of documentation of preeclampsia compared to traditional risk factors for CVD (smoking, diabetes, and chronic hypertension) by reviewing the well-woman visit notes, past medical history, obstetric history, and the problem list in the electronic medical record. For intervention, educational teaching sessions (presentation with Q&A session) and education slide presentations were given to internal medicine physicians at clinic sites. Changes in documentation were evaluated post-intervention. RESULTS: When assessment of relevant pregnancy history was obtained, 23.6% of women were asked about a history preeclampsia while 98.9% were asked about diabetes or smoking and 100% were asked about chronic hypertension (p < 0.001). Education interventions did not significantly change rates of screening documentation (p = 0.36). CONCLUSION: Our study adds to the growing body of literature that women with a history of preeclampsia might not be identified as having increased CVD risk in the outpatient primary care setting. Novel educational programming may be required to increase provider documentation of preeclampsia history in screening.

Ascorbate attenuates red light mediated vasodilation: Potential role of S-nitrosothiols.

There is significant therapeutic advantage of nitric oxide synthase (NOS) independent nitric oxide (NO) production in maladies where endothelium, and thereby NOS, is dysfunctional. Electromagnetic radiation in the red and near infrared region has been shown to stimulate NOS-independent but NO-dependent vasodilation, and thereby has significant therapeutic potential. We have recently shown that red light induces acute vasodilatation in the pre-constricted murine facial artery via the release of an endothelium derived substance. In this study we have investigated the mechanism of vasodilatation and conclude that 670 nm light stimulates vasodilator release from an endothelial store, and that this vasodilator has the characteristics of an S-nitrosothiol (RSNO). This study shows that 670 nm irradiation can be used as a targeted and non-invasive means to release biologically relevant amounts of vasodilator from endothelial stores. This raises the possibility that these stores can be pharmacologically built-up in pathological situations to improve the efficacy of red light treatment. This strategy may overcome eNOS dysfunction in peripheral vascular pathologies for the improvement of vascular health.

Wavelength-dependence of vasodilation and NO release from S-nitrosothiols and dinitrosyl iron complexes by far red/near infrared light.

Far red/near infrared (R/NIR) energy is a novel therapy, but its mechanism of action is poorly characterized. Cytochrome c oxidase (Cco) of the mitochondrial electron transport chain is considered the primary photoacceptor for R/NIR to photolyze a putative heme nitrosyl in Cco to liberate free nitric oxide (NO). We previously observed R/NIR light directly liberates NO from nitrosylated hemoglobin and myoglobin, and recently suggested S-nitrosothiols (RSNO) and dinitrosyl iron complexes (DNIC) may be primary sources of R/NIR-mediated NO. Here we indicate R/NIR light exposure induces wavelength dependent dilation of murine facial artery, with longer wavelengths (740, and 830 nm) exhibiting reduced potency when compared to 670 nm. R/NIR also stimulated NO release from pure solutions of low molecular weight RSNO (GSNO and SNAP) and glutathione dinitrosyl iron complex (GSH-DNIC) in a power- and wavelength-dependent manner, with the greatest effect at 670 nm. NO release from SNAP using 670 was nearly ten-fold more than GSNO or GSH-DNIC, with no substantial difference in NO production at 740 nm and 830 nm. Thermal effects of irradiation on vasodilation or NO release from S-nitrosothiols and DNIC was minimal. Our results suggest 670 nm is the optimal wavelength for R/NIR treatment of certain vascular-related diseases.

Red/near infrared light stimulates release of an endothelium dependent vasodilator and rescues vascular dysfunction in a diabetes model.

Peripheral artery disease (PAD) is a morbid condition whereby ischemic peripheral muscle causes pain and tissue breakdown. Interestingly, PAD risk factors, e.g. diabetes mellitus, cause endothelial dysfunction secondary to decreased nitric oxide (NO) levels, which could explain treatment failures. Previously, we demonstrated 670nm light (R/NIR) increased NO from nitrosyl-heme stores, therefore we hypothesized R/NIR can stimulate vasodilation in healthy and diabetic blood vessels. Vasodilation was tested by ex vivo pressure myography in wild type C57Bl/6, endothelial nitric oxide synthase (eNOS) knockout, and db/db mice (10mW/cm2 for 5min with 10min dark period). NOS inhibition with N-Nitroarginine methyl ester (L-NAME) or the NO scavenger Carboxy-PTIO (c-PTIO) tested the specificity of NO production. 4,5-Diaminofluorescein diacetate (DAF-2) measured NO in human dermal microvascular endothelial cells (HMVEC-d). R/NIR significantly increased vasodilation in wild type and NOS inhibited groups, however R/NIR dilation was totally abolished with c-PTIO and blood vessel denudation. Interestingly, the bath solution from intact R/NIR stimulated vessels could dilate light naïve vessels in a NO dependent manner. Characterization of the bath identified a NO generating substance suggestive of S-nitrosothiols or non heme iron nitrosyl complexes. Consistent with the finding of an endothelial source of NO, intracellular NO increased with R/NIR in HMVEC-d treated with and without L-NAME (1mM), yet c-PTIO (100µm) reduced NO production. R/NIR significantly dilated db/db blood vessels. In conclusion, R/NIR stimulates vasodilation by release of NO bound substances from the endothelium. In a diabetes model of endothelial dysfunction, R/NIR restores vasodilation, which lends the potential for new treatments for diabetic vascular disease.

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice.

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides. IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 ± 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 µg/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 µg/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

An IRF5 Decoy Peptide Reduces Myocardial Inflammation and Fibrosis and Improves Endothelial Cell Function in Tight-Skin Mice.

Interferon regulatory factor 5 (IRF5) has been called a "master switch" for its ability to determine whether cells mount proinflammatory or anti-inflammatory responses. Accordingly, IRF5 should be an attractive target for therapeutic drug development. Here we report on the development of a novel decoy peptide inhibitor of IRF5 that decreases myocardial inflammation and improves vascular endothelial cell (EC) function in tight-skin (Tsk/+) mice. Biolayer interferometry studies showed the Kd of IRF5D for recombinant IRF5 to be 3.72 ± 0.74x10-6M. Increasing concentrations of IRF5D (0-100 µg/mL, 24h) had no significant effect on EC proliferation or apoptosis. Treatment of Tsk/+ mice with IRF5D (1mg/kg/d subcutaneously, 21d) reduced IRF5 and ICAM-1 expression and monocyte/macrophage and neutrophil counts in Tsk/+ hearts compared to expression in hearts from PBS-treated Tsk/+ mice (p<0.05). EC-dependent vasodilatation of facialis arteries isolated from PBS-treated Tsk/+ mice was reduced (~15%). IRF5D treatments (1mg/kg/d, 21d) improved vasodilatation in arteries isolated from Tsk/+ mice nearly 3-fold (~45%, p<0.05), representing nearly 83% of the vasodilatation in arteries isolated from C57Bl/6J mice (~55%). IRF5D (50µg/mL, 24h) reduced nuclear translocation of IRF5 in myocytes cultured on both Tsk/+ cardiac matrix and C57Bl/6J cardiac matrix (p<0.05). These data suggest that IRF5 plays a causal role in inflammation, fibrosis and impaired vascular EC function in Tsk/+ mice and that treatment with IRF5D effectively counters IRF5-dependent mechanisms of inflammation and fibrosis in the myocardium in these mice.