Photobiomodulation alters the viability of HUVECs cells.

The aim of the present study was to investigate the influence of low-level red (660 nm) and infrared (780 nm) laser with four different radiance exposures on human umbilical vein endothelial cells (HUVECs) in vitro. HUVECs (1.5 × 104) were incubated in 96-well culture plates. The cells were maintained in M199 medium supplemented with 20% fetal bovine serum, 1% antibiotic (penicillin), 1% anti-mycotic (Fungizone), and 1% endothelial cell growth supplement. After centrifugation, irradiations (660/780 nm, 40 mW, 1, 5, 10, and 20 J/cm2, 1 s, 5 s, 10 s, and 20 s, respectively, total energy 0.4 J, 2 J, 4 J, and 8 J, and beam spot size at target 0.04 cm2) were performed at the bottom of Falcon tubes such that the laser beam directly reached the cell without passing through the culture medium. The cells were divided into groups based on radiant exposures. Cell viability and protein concentration were verified after 1, 2, 3, 6, 8, and 10 days. Red laser increased the cell viability and protein concentration in all groups (three-way ANOVA, p < 0.05) beginning on the second day. The greatest peak compared with the control was found when the radiant exposure was 5 J/cm2 and 10 J/cm2. Infrared laser inhibited cell viability and modulated the protein concentration in the cells, with the highest peak protein concentration found on the second day in the group with radiant exposure of 1 J/cm2 and 10 J/cm2 (three-way ANOVA, p < 0.05). Red laser increased the viability and concentration of total proteins in HUVECs, whereas infrared laser had an inhibitory effect on cell viability, while maintaining the total protein concentration similar to that found in the control group.

Low-level laser therapy induces human umbilical vascular endothelial cell proliferation, migration and tube formation through activating the PI3K/Akt signaling pathway.

Low-level laser therapy (LLLT) has been recognized as a light therapy that may be used for tissue regeneration, inflammation reduction, and pain relief. We intended to evaluate the effects of LLLT on the proliferation, migration, and tube formation of HUVECs as well as their related mechanisms. HUVECs were exposed to laser irradiation under different laser parameters (irradiation dose, interval and power intensity) in order to choose the optimal parameters, which were determined by the increase in proliferation of HUVECs as follows: irradiation dose of 4.0 J/m2, interval time of 12 h and 6 times in total. The HUVEC proliferation, migration, and tube formation, and levels of angiogenesis-related genes (HIF-1α, eNOS and VEGFA) were examined following LLLT. As suggested by the obtained data, LLLT (1.0, 2.0 and 4.0 J/m2) increased the HUVEC proliferation, migration, and tube formation in dose-and time-dependent manner, accompanied with increases in the levels of HIF-1α, eNOS, and VEGFA. Furthermore, the regulatory mechanism regarding the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway was explored, phosphorylation levels of PI3K and Akt proteins were assessed by Western blot assay, which showed the enhancement of phosphorylation of PI3K, Akt, and mTOR by LLLT. The inhibitor for the PI3K/Akt axis was used to verify the involvement of PI3K/Akt signaling pathway. The obtained results suggested that the inhibition of the PI3K/Akt signaling pathway attenuated the effects of LLLT on proliferation, migration, and angiogenesis of HUVECs. In conclusion, LLLT promotes the proliferation, migration, and angiogenesis of HUVECs via activation of the PI3K/Akt signaling pathway.

Targeting of prostate-specific membrane antigen for radio-ligand therapy of triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer has extremely high risk of relapse due to the lack of targeted therapies, intra- and inter-tumoral heterogeneity, and the inherent and acquired resistance to therapies. In this study, we evaluate the potential of prostate-specific membrane antigen (PSMA) as target for radio-ligand therapy (RLT). METHODS: Tube formation was investigated after incubation of endothelial HUVEC cells in tumor-conditioned media and monitored after staining using microscopy. A binding study with 68Ga-labeled PSMA-addressing ligand was used to indicate targeting potential of PSMA on tumor-conditioned HUVEC cells. For mimicking of the therapeutic application, tube formation potential and vitality of tumor-conditioned HUVEC cells were assessed following an incubation with radiolabeled PSMA-addressing ligand [177Lu]-PSMA-617. For in vivo experiments, NUDE mice were xenografted with triple-negative breast cancer cells MDA-MB231 or estrogen receptor expressing breast cancer cells MCF-7. Biodistribution and binding behavior of [68Ga]-PSMA-11 was investigated in both tumor models at 30 min post injection using µPET. PSMA- and CD31-specific staining was conducted to visualize PSMA expression and neovascularization in tumor tissue ex vivo. RESULTS: The triple-negative breast cancer cells MDA-MB231 showed a high pro-angiogenetic potential on tube formation of endothelial HUVEC cells. The induced endothelial expression of PSMA was efficiently addressed by radiolabeled PSMA-specific ligands. 177Lu-labeled PSMA-617 strongly impaired the vitality and angiogenic potential of HUVEC cells. In vivo, as visualized by µPET, radiolabeled PSMA-ligand accumulated specifically in the triple-negative breast cancer xenograft MDA-MB231 (T/B ratio of 43.3 ± 0.9), while no [68Ga]-PSMA-11 was detected in the estrogen-sensitive MCF-7 xenograft (T/B ratio of 1.1 ± 0.1). An ex vivo immunofluorescence analysis confirmed the localization of PSMA on MDA-MB231 xenograft-associated endothelial cells and also on TNBC cells. CONCLUSIONS: Here we demonstrate PSMA as promising target for two-compartment endogenous radio-ligand therapy of triple-negative breast cancer.

Photobiomodulation Enhances the Angiogenic Effect of Mesenchymal Stem Cells to Mitigate Radiation-Induced Enteropathy.

Radiation-induced enteropathy remains a major complication after accidental or therapeutic exposure to ionizing radiation. Recent evidence suggests that intestinal microvascular damage significantly affects the development of radiation enteropathy. Mesenchymal stem cell (MSC) therapy is a promising tool to regenerate various tissues, including skin and intestine. Further, photobiomodulation (PBM), or low-level light therapy, can accelerate wound healing, especially by stimulating angiogenesis, and stem cells are particularly susceptible to PBM. Here, we explored the effect of PBM on the therapeutic potential of MSCs for the management of radiation enteropathy. In vitro, using human umbilical cord blood-derived MSCs, PBM increased proliferation and self-renewal. Intriguingly, the conditioned medium from MSCs treated with PBM attenuated irradiation-induced apoptosis and impaired tube formation in vascular endothelial cells, and these protective effects were associated with the upregulation of several angiogenic factors. In a mouse model of radiation-induced enteropathy, treatment with PBM-preconditioned MSCs alleviated mucosal destruction, improved crypt cell proliferation and epithelial barrier functions, and significantly attenuated the loss of microvascular endothelial cells in the irradiated intestinal mucosa. This treatment also significantly increased angiogenesis in the lamina propria. Together, we suggest that PBM enhances the angiogenic potential of MSCs, leading to improved therapeutic efficacy for the treatment of radiation-induced enteropathy.

Photobiomodulation (PBM) promotes angiogenesis in-vitro and in chick embryo chorioallantoic membrane model.

The application of light in various therapeutic settings known as Photobiomodulation (PBM) is well established. Indications are the improvement of wound healing and tissue regeneration, scarring, and perfusion as well as pain therapy. Tissue perfusion is mandatory for successful wound healing. Nevertheless, there is a lack of mechanistic studies. We investigate the potential effect of PBM from light emitting diodes (LED) at 635 nm, 80 mW/cm2, 24 J/cm2 on angiogenesis in a two-part study: 1.) Investigation of the effect of PBM on the proliferation of endothelial cells and on vasculogenesis in a co-culture model of endothelial cells and stem cells. 2.) Investigation of the influence of PBM at chick egg chorioallantoic membrane (CAM) assays with fresh human skin xenografts. In both study phases, we observed a stimulating effect of PBM at 635 nm; in part 1: for proliferation of HUVEC (human umbilical vein endothelial cells) (25833 ± 12859 versus 63002 ± 35760 cells/well, p < 0.05, for cellular network formation (2.1 ± 2.1 versus 4.6 ± 3.5, p < 0.05) and for less cell compactness p = 0.01; in part 2: for the increase of number of vessel junctions per ROI (region of interest) (15.9 ± 2.6 versus 20.8 ± 5.4, p < 0.05). Our results suggest significant promotion of angiogenesis by PBM at 635 nm in vitro and in vivo.

Low-level laser irradiation modifies the effect of hyperglycemia on adhesion molecule levels.

Endothelium plays a key role in maintaining vascular homeostasis by secreting active factors involved in many biological processes such as hemostasis, angiogenesis, and inflammation. Hyperglycemia in diabetic patients causes dysfunction of endothelial cells. Soluble fractions of adhesion molecules like sE-selectin and vascular cell adhesion molecule (sVCAM) are considered as markers of endothelial damage. The low-level laser therapy (LLLT) effectively supports the conventional treatment of vascular complications in diabetes, for example hard-to-heal wounds in patients with diabetic foot syndrome. The aim of our study was to evaluate the effect of low-energy laser at the wavelength of 635 nm (visible light) and 830 nm (infrared) on the concentration of adhesion molecules: sE-selectin and sVCAM in the supernatant of endothelial cell culture of HUVEC line. Cells were cultured under high-glucose conditions of 30 mM/L. We have found an increase in sE-selectin and sVCAM levels in the supernatant of cells cultured under hyperglycemic conditions. This fact confirms detrimental influence of hyperglycemia on vascular endothelial cell cultures. LLLT can modulate the inflammation process. It leads to a decrease in sE-selectin and sVCAM concentration in the supernatant and an increase in the number of endothelial cells cultured under hyperglycemic conditions. The influence of LLLT is greater at the wavelength of 830 nm.

Low-level laser irradiation at a high power intensity increased human endothelial cell exosome secretion via Wnt signaling.

The distinct role of low-level laser irradiation (LLLI) on endothelial exosome biogenesis remains unclear. We hypothesize that laser irradiation of high dose in human endothelial cells (ECs) contributes to the modulation of exosome biogenesis via Wnt signaling pathway. When human ECs were treated with LLLI at a power density of 80 J/cm2, the survival rate reduced. The potential of irradiated cells to release exosomes was increased significantly by expressing genes CD63, Alix, Rab27a, and b. This occurrence coincided with an enhanced acetylcholine esterase activity, pseudopodia formation, and reduced zeta potential value 24 h post-irradiation. Western blotting showed the induction of LC3 and reduced level of P62, confirming autophagy response. Flow cytometry and electron microscopy analyses revealed the health status of the mitochondrial function indicated by normal ΔΨ activity without any changes in the transcription level of PINK1 and Optineurin. When cells exposed to high power laser irradiation, p-Akt/Akt ratio and in vitro tubulogenesis capacity were blunted. PCR array and bioinformatics analyses showed the induction of transcription factors promoting Wnt signaling pathways and GTPase activity. Thus, LLLI at high power intensity increased exosome biogenesis by the induction of autophagy and Wnt signaling. LLLI at high power intensity increases exosome biogenesis by engaging the transcription factors related to Wnt signaling and autophagy stimulate.

Salidroside, a scavenger of ROS, enhances the radioprotective effect of Ex-RAD® via a p53-dependent apoptotic pathway.

Salidroside (Sal), the predominant component of a Chinese medicinal herb, Rhodiola rosea L., has become an attractive bioagent due to its significant anti-radiation, antioxidant and immune adjustment effects. We explored the radioprotective effect of Sal to ascertain whether it could enhance the anti-radiation effect of ON 01210.Na (Ex-RAD®) in vivo and in vitro, and elucidate its underlying mechanism. Our data demonstrated that Sal inhibited radiation-induced apoptosis, scavenged reactive oxygen species (ROS), and decreased the DNA damage of human umbilical vein endothelial cells (HUVECs). Sal downregulated the expression of Bax and p53 and increased the ratio of Bcl-2/Bax, which indicated that Sal inhibited the radiation-induced apoptosis through p53-dependent pathways. The radioprotection of the Sal pretreatment was also evidenced by an increasing survival rate of the mice, maintaining antioxidant enzyme levels in the liver, and accelerating hematopoietic recovery. The results suggest that Sal exhibits an excellent radioprotective effect with powerful antioxidant activity in vitro and in vivo. Sal enhanced the radioprotective effect of Ex-RAD by improving the antioxidant effect, the scavenging of ROS, by accelerating hematopoietic recovery and DNA repair as well as by regulating apoptotic and repair signaling pathways. Combined modality treatments were more effective than single-agent treatments, demonstrating the value of multiple-agent radioprotectants.

Effects of combination of melatonin and laser irradiation on ovarian cancer cells and endothelial lineage viability.

The main goal of anti-cancer therapeutic approaches is to induce apoptosis in tumor masses but not in the normal tissues. Nevertheless, the combination of photodynamic irradiation with complementary oncostatic agents contributes to better therapeutic performance. Here, we applied two different cell lines; SKOV3 ovarian carcinoma cells and HUVECs umbilical cord cells as in vitro models to pinpoint whether pharmacological concentration of melatonin in combination with photodynamic therapy induces cell cytotoxicity. The cells were separately treated with various concentrations of melatonin (0 to 10 mM) and photodynamic irradiation alone or in combination. Cells were preliminary exposed to increasing concentrations of melatonin for 24 h and subsequently underwent laser irradiation for 60 s with an output power of 80 mW in continuous mode at 675 nm wavelength and a total light dose of 13.22 J/cm2. Cell viability, apoptosis/necrosis rates, and reactive oxygen species levels as well as heat shock protein 70 expression were monitored after single and combined treatments. A statistical analysis was performed by applying one-way analysis of variance (ANOVA) and post hoc Tukey's test. Combination treatment of both cell lines caused a marked increase in apoptosis/necrosis rate, reactive oxygen species generation, and heat shock protein 70 expression compared to incubation of the cells with each agent alone (p < 0.05). SKOV3 cancer cells expressed higher level of heat shock protein 70 under experimental procedure as compared to HUVECs (p < 0.05). Our results introduce melatonin as a potent stimulus for enhancing the efficacy of laser on induction of apoptosis in tumor cells.

In vitro studies on the mechanisms involved in chemoprevention using Calluna vulgaris on vascular endothelial cells exposed to UVB.

The study aims to investigate the mechanisms involved in the in vitro effect of UVB on endothelial vascular cells (HUVECs) pretreated with a photochemopreventive agent, the Calluna vulgaris (Cv) extract. Two concentrations of Cv, below the limit of cytotoxicity IC50 (2.5 and 7.5 µg GAE/ml) and two doses of UVB (50 and 100 mJ/cm(2)) were used. Oxidative stress parameters were quantified at 1 h and 24 h after irradiation and apoptosis, DNA damage and the induction/activation of NF-κB were evaluated at 24 h. UVB exposure led to the formation of lipid peroxides in a dose dependent manner (p<0.001), induced apoptosis, increased the γ-H2AX levels and the activation of NF-κB. Pretreatment with 2.5 µg GAE/ml Cv improved the antioxidant defense, protected against DNA lesions and was able to decrease cellular death at low dose of irradiation. 7.5 µg GAE/ml Cv was prooxidant, favored the formation of DNA lesions, amplified the NF-κB activation UVB-induced (p<0.01) and led to high levels of cellular death. Both doses of Cv inhibited caspase-3 activation. The modulatory effect of Cv extract on endothelial cells exposed to UVB depend on the concentration of Cv used. This study provides insides into the mechanisms triggered by UVB and antioxidants on skin endothelial cells.

Phototherapy with low-level laser influences the proliferation of endothelial cells and vascular endothelial growth factor and transforming growth factor-beta secretion.

The healing process and the angiogenesis associated with it, is a very important but currently poorly understood area. Low level laser therapy (LLLT) has been reported to modulate the process of tissue repair by stimulation of cellular reaction such as migration, proliferation, apoptosis and cellular differentiation. The aim of this work was to evaluate the influence of laser radiation in the range of visible and infrared light on the proliferation of vascular endothelial cells in vitro and the secretion of angiogenic factors: vascular endothelial growth factor (VEGF)-A and transforming growth factor (TGF)-ß. Vascular human endothelial cells (Ecs) were exposed to radiation with laser beam of the wavelengths: 635 nm (1.875 mW/cm²) and 830 nm (3.75 mW/cm²). Depending on the radiation energy density, the experiment was conducted in four groups : I) the control group (no radiation, 0 J/cm²); II) 635 nm - the energy density was 2 J/cm²; III) 635 nm - 4 J/cm²; IV635 nm - 8 J/cm², II) 830 nm - the energy density was 2 J/cm²; III) 830 nm - 4 J/cm²; IV) 830 nm - 8 J/cm². The proliferation and concentration of VEGF-A and TGF-ß were examined. LLLT with wavelength 635 nm increases endothelial cell proliferation. Significant increase in endothelial cell proliferation and corresponding decrease in VEGF concentration may suggest the role for VEGF in this process. The wavelength of 830 nm was associated with a decrease in TGF-ß secretion.

Far red/near infrared light treatment promotes femoral artery collateralization in the ischemic hindlimb.

Nitric oxide (NO) is a crucial mediator of hindlimb collateralization and angiogenesis. Within tissues there are nitrosyl-heme proteins which have the potential to generate NO under conditions of hypoxia or low pH. Low level irradiation of blood and muscle with light in the far red/near infrared spectrum (670 nm, R/NIR) facilitates NO release. Therefore, we assessed the impact of red light exposure on the stimulation of femoral artery collateralization. Rabbits and mice underwent unilateral resection of the femoral artery and chronic R/NIR treatment. The direct NO scavenger carboxy-PTIO and the nitric oxide synthase (NOS) inhibitor L-NAME were also administered in the presence of R/NIR. DAF fluorescence assessed R/NIR changes in NO levels within endothelial cells. In vitro measures of R/NIR induced angiogenesis were assessed by endothelial cell proliferation and migration. R/NIR significantly increased collateral vessel number which could not be attenuated with L-NAME. R/NIR induced collateralization was abolished with c-PTIO. In vitro, NO production increased in endothelial cells with R/NIR exposure, and this finding was independent of NOS inhibition. Similarly R/NIR induced proliferation and tube formation in a NO dependent manner. Finally, nitrite supplementation accelerated R/NIR collateralization in wild type C57Bl/6 mice. In an eNOS deficient transgenic mouse model, R/NIR restores collateral development. In conclusion, R/NIR increases NO levels independent of NOS activity, and leads to the observed enhancement of hindlimb collateralization.

Proteomic analysis by SILAC and 2D-DIGE reveals radiation-induced endothelial response: four key pathways.

Epidemiological data show that ionising radiation increases the risk of cardiovascular disease. The endothelium is one of the main targets of radiation-induced damage. Rapid radiation-induced alterations in the biological processes were investigated after exposure to a clinically relevant radiation dose (2.5 Gy gamma radiation). The changes in protein expression were determined using the human endothelial cell line EA.hy926 as a model. Two complementary proteomic approaches, SILAC (Stable Isotope Labelling with Amino acids in Cell culture) and 2D-DIGE (Two Dimensional Difference-in-Gel-Electrophoresis) were used. The proteomes of the endothelial cells were analysed 4h and 24h after irradiation. Differentially expressed proteins were identified and quantified by MALDI-TOF/TOF and LTQ Orbitrap tandem mass spectrometry. The deregulated proteins were mainly categorised in four key pathways: (i) glycolysis/gluconeogenesis and synthesis/degradation of ketone bodies, (ii) oxidative phosphorylation, (iii) Rho-mediated cell motility and (iv) non-homologous end joining. We suggest that these alterations facilitate the repair processes needed to overcome the stress caused by irradiation and are indicative of the vascular damage leading to radiation-induced cardio- and cerebrovascular impairment.

Far-infrared therapy induces the nuclear translocation of PLZF which inhibits VEGF-induced proliferation in human umbilical vein endothelial cells.

Many studies suggest that far-infrared (FIR) therapy can reduce the frequency of some vascular-related diseases. The non-thermal effect of FIR was recently found to play a role in the long-term protective effect on vascular function, but its molecular mechanism is still unknown. In the present study, we evaluated the biological effect of FIR on vascular endothelial growth factor (VEGF)-induced proliferation in human umbilical vein endothelial cells (HUVECs). We found that FIR ranging 3∼10 µm significantly inhibited VEGF-induced proliferation in HUVECs. According to intensity and time course analyses, the inhibitory effect of FIR peaked at an effective intensity of 0.13 mW/cm(2) at 30 min. On the other hand, a thermal effect did not inhibit VEGF-induced proliferation in HUVECs. FIR exposure also inhibited the VEGF-induced phosphorylation of extracellular signal-regulated kinases in HUVECs. FIR exposure further induced the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and NO generation in VEGF-treated HUVECs. Both VEGF-induced NO and reactive oxygen species generation was involved in the inhibitory effect of FIR. Nitrotyrosine formation significantly increased in HUVECs treated with VEGF and FIR together. Inhibition of phosphoinositide 3-kinase (PI3K) by wortmannin abolished the FIR-induced phosphorylation of eNOS and Akt in HUVECs. FIR exposure upregulated the expression of PI3K p85 at the transcriptional level. We further found that FIR exposure induced the nuclear translocation of promyelocytic leukemia zinc finger protein (PLZF) in HUVECs. This induction was independent of a thermal effect. The small interfering RNA transfection of PLZF blocked FIR-increased PI3K levels and the inhibitory effect of FIR. These data suggest that FIR induces the nuclear translocation of PLZF which inhibits VEGF-induced proliferation in HUVECs.