Secretome from iPSC-derived MSCs exerts proangiogenic and immunosuppressive effects to alleviate radiation-induced vascular endothelial cell damage.

BACKGROUND: Radiation therapy is the standard of care for central nervous system tumours. Despite the success of radiation therapy in reducing tumour mass, irradiation (IR)-induced vasculopathies and neuroinflammation contribute to late-delayed complications, neurodegeneration, and premature ageing in long-term cancer survivors. Mesenchymal stromal cells (MSCs) are adult stem cells that facilitate tissue integrity, homeostasis, and repair. Here, we investigated the potential of the iPSC-derived MSC (iMSC) secretome in immunomodulation and vasculature repair in response to radiation injury utilizing human cell lines. METHODS: We generated iPSC-derived iMSC lines and evaluated the potential of their conditioned media (iMSC CM) to treat IR-induced injuries in human monocytes (THP1) and brain vascular endothelial cells (hCMEC/D3). We further assessed factors in the iMSC secretome, their modulation, and the molecular pathways they elicit. RESULTS: Increasing doses of IR disturbed endothelial tube and spheroid formation in hCMEC/D3. When IR-injured hCMEC/D3 (IR ≤ 5 Gy) were treated with iMSC CM, endothelial cell viability, adherence, spheroid compactness, and proangiogenic sprout formation were significantly ameliorated, and IR-induced ROS levels were reduced. iMSC CM augmented tube formation in cocultures of hCMEC/D3 and iMSCs. Consistently, iMSC CM facilitated angiogenesis in a zebrafish model in vivo. Furthermore, iMSC CM suppressed IR-induced NFκB activation, TNF-α release, and ROS production in THP1 cells. Additionally, iMSC CM diminished NF-kB activation in THP1 cells cocultured with irradiated hCMEC/D3, iMSCs, or HMC3 microglial lines. The cytokine array revealed that iMSC CM contains the proangiogenic and immunosuppressive factors MCP1/CCL2, IL6, IL8/CXCL8, ANG (Angiogenin), GROα/CXCL1, and RANTES/CCL5. Common promoter regulatory elements were enriched in TF-binding motifs such as androgen receptor (ANDR) and GATA2. hCMEC/D3 phosphokinome profiling revealed increased expression of pro-survival factors, the PI3K/AKT/mTOR modulator PRAS40 and ß-catenin in response to CM. The transcriptome analysis revealed increased expression of GATA2 in iMSCs and the enrichment of pathways involved in RNA metabolism, translation, mitochondrial respiration, DNA damage repair, and neurodevelopment. CONCLUSIONS: The iMSC secretome is a comodulated composite of proangiogenic and immunosuppressive factors that has the potential to alleviate radiation-induced vascular endothelial cell damage and immune activation.

Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation.

Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.

Bone marrow coagulated and low-level laser therapy accelerate bone healing by enhancing angiogenesis, cell proliferation, osteoblast differentiation, and mineralization.

The present study evaluated bone marrow aspirate (BMA) and low-level laser therapy (LLLT) on bone healing. It was created critical-size defects (CSD) of 5 mm diameter in rat calvaria of 64 rats. Animals were randomly divided into four groups: Control (blood clot), BMA (coagulated BMA), LLLT (laser irradiation and blood clot), and BMA/LLLT (laser irradiation and coagulated BMA). Euthanasia was performed at 15 or 30 days postoperative. Immunohistochemical reactions were performed to identify vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA), runt-related transcription factor-2 (Runx2), bone morphogenetic protein-2 (BMP-2), osteocalcin (OCN), and osteopontin (OPN). The markers were quantified, and data were statistically analyzed. Groups BMA/LLLT and LLLT presented significantly higher VEGF expression than group control. Group BMA/LLLT presented a significantly higher expression of PCNA than all experimental groups. Groups BMA and BMA/LLLT presented significantly higher expression of BMP-2 than all experimental groups. Groups LLLT and BMA/LLLT presented significantly higher expression of OPN than groups control and BMA. Groups LLLT, BMA, and BMA/LLLT presented a significantly higher expression of OCN than group control. It can be concluded that the association of BMA and LLLT enhanced bone healing by improving expression of VEGF, PCNA, Runx2, BMP-2, OPN, and OCN.

Development of a Rat Model of Mandibular Irradiation Sequelae for Preclinical Studies of Bone Repair.

Repairing mandibular bone defects after radiotherapy of the upper aerodigestive tract is clinically challenging. Although bone tissue engineering has recently generated a number of innovative treatment approaches for osteoradionecrosis (ORN), these modalities must be evaluated preclinically in a relevant, reproducible, animal model. The objective of this study was to evaluate a novel rat model of mandibular irradiation sequelae, with a focus on the adverse effects of radiotherapy on bone structure, intraosseous vascularization, and bone regeneration. Rats were irradiated with a single 80 Gy dose to the jaws. Three weeks after irradiation, mandibular bone defects of different sizes (0, 1, 3, or 5 mm) were produced in each hemimandible. Five weeks after the surgical procedure, the animals were euthanized. Explanted mandibular samples were qualitatively and quantitatively assessed for bone formation, bone structure, and intraosseous vascular volume by using micro-computed tomography, scanning electron microscopy, and histology. Twenty irradiated hemimandibles and 20 nonirradiated hemimandibles were included in the study. The bone and vessel volumes were significantly lower in the irradiated group. The extent of bone remodeling was inversely related to the defect size. In the irradiated group, scanning electron microscopy revealed a large number of polycyclic gaps consistent with periosteocytic lysis (described as being pathognomonic for ORN). This feature was correlated with elevated osteoclastic activity in a histological assessment. In the irradiated areas, the critical-sized defect was 3 mm. Hence, our rat model of mandibular irradiation sequelae showed hypovascularization and osteopenia. Impact statement Repairing mandibular bone defects after radiotherapy of the upper aerodigestive tract is clinically challenging. Novel tissue engineering approaches for healing irradiated bone must first be assessed in animal models. The current rat model of mandibular irradiation sequelae is based on tooth extraction after radiotherapy. However, the mucosal sequelae of radiotherapy often prevent the retention of tissue-engineered biomaterials within the bone defect. We used a submandibular approach to create a new rat model of mandibular irradiation sequelae, which enables the stable retention of biomaterials within the bone defect and should thus facilitate the assessment of bone regeneration.

Effects of Hypoxia and Radiation-Induced Exosomes on Migration of Lung Cancer Cells and Angiogenesis of Umbilical Vein Endothelial Cells.

Numerous studies have shown that exosomes play important roles in tumor biology development. However, the function of exosomal protein in cancer progression under different oxygen condition after irradiation is poorly understood. In this study, non-small cell lung cancer (NSCLC) A549 cells were γ-ray irradiated under normoxic or hypoxic conditions, then the exosomes released from the irradiated cells were collected and co-cultured with nonirradiated A549 cells or human umbilical vein endothelial cells (HUVECs). It was found that the exosomes significantly promoted the proliferation, migration and invasion of A549 cells as well as the proliferation and angiogenesis of HUVECs. Moreover, the exosomes released from hypoxic cells and/or irradiated cells had more powerful driving force in tumor progression compared to that generated from normoxia cells. Meanwhile, the proteins contained in the exosomes derived from A549 cells under different conditions were detected using tandem mass tag (TMT), and their expression profiles were analyzed. It was found that the exosome-derived protein of angiopoietin-like 4 (ANGPTL4) contributed to the migration of A549 cells as well as the angiogenesis of HUVECs, suggesting its potential as an effective diagnostic biomarker of metastasis and even a therapeutic target of lung cancer.

miR-548aq-3p is a novel target of Far infrared radiation which predicts coronary artery disease endothelial colony forming cell responsiveness.

Non-invasive far infrared radiation (FIR) has been observed to improve the health of patients with coronary artery disease (CAD). Endothelial colony forming cells (ECFCs) contribute to vascular repair and CAD. The goal of this study was to uncover the role of FIR in ECFCs function and to reveal potential biomarkers for indication of FIR therapy in CAD patients. FIR significantly enhanced in vitro migration (transwell assay) and tube formation (tube length) capacities in a subpopulation of CAD ECFCs. Clinical parameters associated with the responsiveness of ECFCs to FIR include smoking and gender. ECFCs from CAD patients that smoke did not respond to FIR in most cases. In contrast, ECFCs from females showed a higher responsiveness to FIR than ECFCs from males. To decipher the molecular mechanisms by which FIR modulates ECFCs functions, regardless of sex, RNA sequencing analysis was performed in both genders of FIR-responsive and FIR-non/unresponsive ECFCs. Gene Ontology (GO) analysis of FIR up-regulated genes indicated that the pathways enriched in FIR-responsive ECFCs were involved in cell viability, angiogenesis and transcription. Small RNA sequencing illustrated 18 and 14 miRNAs that are up- and down-regulated, respectively, in FIR-responsive CAD ECFCs in both genders. Among the top 5 up- and down-regulated miRNAs, down-regulation of miR-548aq-3p in CAD ECFCs after FIR treatment was observed in FIR-responsive CAD ECFCs by RT-qPCR. Down-regulation of miR-548aq-3p was correlated with the tube formation activity of CAD ECFCs enhanced by FIR. After establishment of the down-regulation of miR-548aq-3p by FIR in CAD ECFCs, we demonstrated through overexpression and knockdown experiments that miR-548aq-3p contributes to the inhibition of the tube formation of ECFCs. This study suggests the down-regulation of miR-548aq-3p by FIR may contribute to the improvement of ECFCs function, and represents a novel biomarker for therapeutic usage of FIR in CAD patients.

Low doses of ionizing radiation enhance angiogenesis and consequently accelerate post-embryonic development but not regeneration in zebrafish.

Low doses of ionizing radiation (LDIR) activate endothelial cells inducing angiogenesis. In zebrafish, LDIR induce vessel formation in the sub-intestinal vessels during post-embryonic development and enhance the inter-ray vessel density in adult fin regeneration. Since angiogenesis is a crucial process involved in both post-embryonic development and regeneration, herein we aimed to understand whether LDIR accelerate these physiological conditions. Our data show that LDIR upregulate the gene expression of several pro-angiogenic molecules, such as flt1, kdr, angpt2a, tgfb2, fgf2 and cyr61in sorted endothelial cells from zebrafish larvae and this effect was abrogated by using a vascular endothelial growth factor receptor (VEGFR)-2 tyrosine kinase inhibitor. Irradiated zebrafish present normal indicators of developmental progress but, importantly LDIR accelerate post-embryonic development in a VEGFR-2 dependent signaling. Furthermore, our data show that LDIR do not accelerate regeneration after caudal fin amputation and the gene expression of the early stages markers of regeneration are not modulated by LDIR. Even though regeneration is considered as a recapitulation of embryonic development and LDIR induce angiogenesis in both conditions, our findings show that LDIR accelerate post-embryonic development but not regeneration. This highlights the importance of the physiological context for a specific phenotype promoted by LDIR.

Extracorporeal Shock Wave Stimulates Angiogenesis and Collagen Production in Facial Soft Tissue.

BACKGROUND: This study investigated the efficacy of extracorporeal shock wave (ESW) application in stimulating dermal thickness, vascularity, and collagen synthesis of facial skin in a large animal model. MATERIALS AND METHODS: The facial skin of the maxillary and mandibular areas of goats (n = 6 per group) was treated with ESWs of different intensities (0.15 and 0.45 mJ/mm2; 1000 pulses). After 4 d, histology and immunohistochemistry were used to evaluate the following: dermal thickness, total number and abundance of microvessels, amount of type 1 collagen, and α-smooth muscle actin expression. RESULTS: Dermal thickness, number and abundance of microvessels, and collagen synthesis increased after ESW application at both intensities (each P < 0.05). When comparing ESW groups, the highest collagen abundance was seen after 0.15 mJ/mm2 (P = 0.034), whereas the highest number of microvessels was detected after treatment with 0.45 mJ/mm2 (P = 0.002). CONCLUSIONS: A single-session application of focused low-energy ESWs to facial skin can increase dermal thickness by stimulating collagen production and local microcirculation. These findings commend the technique for future investigation for pretreatment of local or microvascular skin flaps to enhance tissue healing.

Photobiomodulation of red and green lights in the repair process of third-degree skin burns.

The aim of this study was to evaluate the photobiomodulation of red and green lights in the repair process of third-degree skin burns in rats through clinicopathological and immunohistochemical parameters. Sixty male Wistar rats were divided into three groups: control (CTRL) (n = 20), red LED (RED) (n = 20), and green LED (GREEN) (n = 20), with subgroups (n = 5) for each time of euthanasia (7, 14, 21, and 28 days). Daily applications in RED (λ630 ± 10 nm, 300 mW) and GREEN groups (λ520 ± 30 nm, 180 mW) were performed at four points of the wound (total 36 J/cm2 in RED and 240 J/cm2 in GREEN). After euthanasia, the wound retraction index (WRI) was evaluated. In histological sections, the re-epithelialization degree, the angiogenic index (AI), and the amount of myofibroblasts in wounds were analyzed. At 14 and 21 days, the RED group induced higher re-epithelialization and WRI compared to CTRL (p > 0.05) and GREEN groups (p < 0.05). At 7 and 14 days, greater AI were observed in the GREEN group, with significant difference in relation to CTRL group at 7 days (p < 0.05). At 21 and 28 days, a trend was observed for greater amount of myofibroblasts in the GREEN group, with significant difference in relation to CTRL group at 21 days (p < 0.05). The results suggest greater potential of the green light to stimulate angiogenesis in the initial periods and myofibroblastic differentiation in the final periods of the repair of third-degree skin burns. Red light may stimulate further re-epithelialization and wound retraction, especially in advanced repair phases.

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.

Exposure to blue light stimulates the proangiogenic capability of exosomes derived from human umbilical cord mesenchymal stem cells.

BACKGROUND: The therapeutic potential of mesenchymal stem cells (MSCs) may be attributed partly to the secreted paracrine factors, which comprise exosomes. Exosomes are small, saucer-shaped vesicles containing miRNAs, mRNAs, and proteins. Exosomes derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) have been reported to promote angiogenesis. However, the efficacy of exosome-based therapies is still limited both in vitro and in vivo. The present study aimed to develop a new optical manipulation approach to stimulate the proangiogenic potential of exosomes and characterize its mechanism underlying tissue regeneration. METHODS: We used blue (455 nm) and red (638 nm) monochromatic light exposure to investigate the processing of stimuli. Exosomes were prepared by QIAGEN exoEasy Maxi kit and confirmed to be present by transmission electron microscopy and immunoblotting analyses. The proangiogenic activity of blue light-treated human umbilical vein endothelial cells (HUVECs), when co-cultured with hUC-MSCs, was assessed by EdU (5-ethynyl-2'-deoxyuridine) incorporation, wound closure, and endothelial tube formation assays. The in vivo angiogenic activity of blue light-treated MSC-derived exosomes (MSC-Exs) was evaluated using both murine matrigel plug and skin wound models. RESULTS: We found that 455-nm blue light is effective for promoting proliferation, migration, and tube formation of HUVECs co-cultured with MSCs. Furthermore, MSC-Exs stimulated in vivo angiogenesis and their proangiogenic potential were enhanced significantly upon blue light illumination. Finally, activation of the endothelial cells in response to stimulation by blue light-treated exosomes was demonstrated by upregulation of two miRNAs, miR-135b-5p, and miR-499a-3p. CONCLUSIONS: Blue (455 nm) light illumination improved the therapeutic effects of hUC-MSC exosomes by enhancing their proangiogenic ability in vitro and in vivo with the upregulation of the following two miRNAs: miR-135b-5p and miR-499a-3p.

Conjugated polymers optically regulate the fate of endothelial colony-forming cells.

The control of stem and progenitor cell fate is emerging as a compelling urgency for regenerative medicine. Here, we propose a innovative strategy to gain optical control of endothelial colony-forming cell fate, which represents the only known truly endothelial precursor showing robust in vitro proliferation and overwhelming vessel formation in vivo. We combine conjugated polymers, used as photo-actuators, with the advantages offered by optical stimulation over current electromechanical and chemical stimulation approaches. Light modulation provides unprecedented spatial and temporal resolution, permitting at the same time lower invasiveness and higher selectivity. We demonstrate that polymer-mediated optical excitation induces a robust enhancement of proliferation and lumen formation in vitro. We identify the underlying biophysical pathway as due to light-induced activation of TRPV1 channel. Altogether, our results represent an effective way to induce angiogenesis in vitro, which represents the proof of principle to improve the outcome of autologous cell-based therapy in vivo.

Electrical stimulation promotes the angiogenic potential of adipose-derived stem cells.

Autologous fat transfer (AFT) is limited by post-operative volume loss due to ischemia-induced cell death in the fat graft. Previous studies have demonstrated that electrical stimulation (ES) promotes angiogenesis in a variety of tissues and cell types. In this study we investigated the effects of ES on the angiogenic potential of adipose-derived stem cells (ASC), important progenitor cells in fat grafts with proven angiogenic potential. Cultured human ASC were electrically stimulated for 72 hours after which the medium of stimulated (ES) and non-stimulated (control) ASC was analysed for angiogenesis-related proteins by protein array and ELISA. The functional effect of ES on angiogenesis was then assessed in vitro and in vivo. Nine angiogenesis-related proteins were detected in the medium of electrically (non-)stimulated ASC and were quantified by ELISA. The pro-angiogenic proteins VEGF and MCP-1 were significantly increased following ES compared to controls, while the anti-angiogenic factor Serpin E1/PAI-1 was significantly decreased. Despite increased levels of anti-angiogenic TSP-1 and TIMP-1, medium of ES-treated ASC significantly increased vessel density, total vessel network length and branching points in chorio-allantoic membrane assays. In conclusion, our proof-of-concept study showed that ES increased the angiogenic potential of ASC both in vitro and in vivo.

Applications of Ultrasound to Stimulate Therapeutic Revascularization.

Many pathological conditions are characterized or caused by the presence of an insufficient or aberrant local vasculature. Thus, therapeutic approaches aimed at modulating the caliber and/or density of the vasculature by controlling angiogenesis and arteriogenesis have been under development for many years. As our understanding of the underlying cellular and molecular mechanisms of these vascular growth processes continues to grow, so too do the available targets for therapeutic intervention. Nonetheless, the tools needed to implement such therapies have often had inherent weaknesses (i.e., invasiveness, expense, poor targeting, and control) that preclude successful outcomes. Approximately 20 years ago, the potential for using ultrasound as a new tool for therapeutically manipulating angiogenesis and arteriogenesis began to emerge. Indeed, the ability of ultrasound, especially when used in combination with contrast agent microbubbles, to mechanically manipulate the microvasculature has opened several doors for exploration. In turn, multiple studies on the influence of ultrasound-mediated bioeffects on vascular growth and the use of ultrasound for the targeted stimulation of blood vessel growth via drug and gene delivery have been performed and published over the years. In this review article, we first discuss the basic principles of therapeutic ultrasound for stimulating angiogenesis and arteriogenesis. We then follow this with a comprehensive cataloging of studies that have used ultrasound for stimulating revascularization to date. Finally, we offer a brief perspective on the future of such approaches, in the context of both further research development and possible clinical translation.

Stereological and gene expression examinations on the combined effects of photobiomodulation and curcumin on wound healing in type one diabetic rats.

We examined the effects of photobiomodulation (PBM) independently and combined with curcumin on stereological parameters and basic fibroblast growth factor (bFGF), hypoxia-inducible factor-1α (HIF-1α), and stromal cell-derived factor-1α (SDF-1α) gene expressions in an excisional wound model of rats with type one diabetes mellitus (T1DM). T1DM was induced by an injection of streptozotocin (STZ) in each of the 90 male Wistar rats. One round excision was generated in the skin on the back of each of the 108 rats. The rats were divided into six groups (n = 18 per group): control (diabetic), untreated group; vehicle (diabetic) group, which received sesame oil; PBM (diabetic) group; curcumin (diabetic) group; PBM + curcumin (diabetic) group; and a healthy control group. On days 4, 7, and 15, we conducted both stereological and quantitative real-time PCR (qRT-PCR) analyses. The PBM and PBM + curcumin groups had significantly better inflammatory response modulation in terms of macrophages (P < .01), neutrophils (P < .001), and increased fibroblast values compared with the other groups at day 4 (P < .001), day 7 (P < .01), and day 15 (P < .001). PBM treatment resulted in increased bFGF gene expression on days 4 (P < .001) and 7 (P < .001), and SDF-1α gene expression on day 4 (P < .001). The curcumin group had increased bFGF (P < .001) expression on day 4. Both the PBM and PBM + curcumin groups significantly increased wound healing by modulation of the inflammatory response, and increased fibroblast values and angiogenesis. The PBM group increased bFGF and SDF-1α according to stereological and gene expression analyses compared with the other groups. The PBM and PBM + curcumin groups significantly increased the skin injury repair process to more rapidly reach the proliferation phase of the wound healing in T1DM rats.

Synechococcus elongatus PCC7942 secretes extracellular vesicles to accelerate cutaneous wound healing by promoting angiogenesis.

Poor wound healing affects millions of people worldwide each year and needs better therapeutic strategies. Synechococcus elongatus PCC 7942 is a naturally occurring photoautotrophic cyanobacterium that can be easily obtained and large-scale expanded. Here, we investigated the therapeutic efficacy of this cyanobacterium in a mouse model of acute burn injury and whether the secretion of extracellular vesicles (EVs), important mediators of cell paracrine activity, is a key mechanism of the cyanobacterium-induced regulation of wound healing. Methods: The effects of Synechococcus elongatus PCC 7942 on burn wound healing in mice under light or dark conditions were evaluated by measuring wound closure rates, histological and immunofluorescence analyses. A series of assays in vivo and in vitro were conducted to assess the impact of the cyanobacterium on angiogenesis. GW4869 was used to interfere with the secretion of EVs by the cyanobacterium and the abilities of the GW4869-pretreated and untreated Synechococcus elongatus PCC 7942 to regulate endothelial angiogenesis were compared. The direct effects of the cyanobacterium-derived EVs (S. elongatus-EVs) on angiogenesis, wound healing and expressions of a class of pro-inflammatory factors that have regulatory roles in wound healing were also examined. Results: Synechococcus elongatus PCC 7942 treatment under light and dark conditions both significantly promoted angiogenesis and burn wound repair in mice. In vitro, the cyanobacterium enhanced angiogenic activities of endothelial cells, but the effects were markedly blocked by GW4869 pretreatment. S. elongatus-EVs were capable of augmenting endothelial angiogenesis in vitro, and stimulating new blood vessel formation and burn wound healing in mice. The expression of interleukin 6 (IL-6), which has an essential role in angiogenesis during skin wound repair, was induced in wound tissues and wound healing-related cells by S. elongatus-EVs and Synechococcus elongatus PCC 7942. Conclusion: Synechococcus elongatus PCC 7942 has the potential as a promising strategy for therapeutic angiogenesis and wound healing primarily by the delivery of functional EVs, not by its photosynthetic activity. The promotion of IL-6 expression may be a mechanism of the cyanobacterium and its EVs-induced pro-angiogenic and -wound healing effects.

Low-level laser therapy affects dentinogenesis and angiogenesis of in vitro 3D cultures of dentin-pulp complex.

To investigate the effects of gallium-aluminum-arsenide (GaAlAs) diode laser low-level laser therapy (LLLT) on angiogenesis and dentinogenesis of the dentin-pulp complex in a human tooth slice-based in vitro model. Forty tooth slices were prepared from 31 human third molars. Slices were cultured at 37 °C, 5% CO2, and 95% humidity and randomly assigned to one of the following groups: group I: no laser treatment, group II: 660-nm diode laser; energy density = 1 J/cm2, group III: 660-nm diode laser; energy density = 3 J/cm2, group IV: 810-nm diode laser; energy density = 1 J/cm2 and group V: 810-nm diode laser; energy density = 3 J/cm2. LLLT was applied on the third and fifth days of culture. After 7 days, tissues were retrieved for real-time RT-PCR analysis to investigate the expression of VEGF, VEGFR2, DSPP, DMP-1, and BSP in respect to controls. Lower energy density (1 J/cm2) with the 660 nm wavelength showed a statistically significant up-regulation of both angiogenic (VEGF: 15.3-folds and VEGFR2: 3.8-folds) and odontogenic genes (DSPP: 6.1-folds, DMP-1: 3-fold, and BSP: 6.7-folds). While the higher energy density (3 J/cm2) with the 810 nm wavelength resulted in statistically significant up-regulation of odontogenic genes (DSPP: 2.5-folds, DMP-1: 17.7-folds, and BSP: 7.1-folds), however, the angiogenic genes had variable results where VEGF was up-regulated while VEGFR2 was down-regulated. Low-level laser therapy could be a useful tool to promote angiogenesis and dentinogenesis of the dentin-pulp complex when parameters are optimized.

Irradiation Delays Tissue Growth but Enhances Osteogenic Differentiation in Vascularized Constructs.

BACKGROUND: Regenerative medicine is still deficient in the reconstruction after cancer due to impaired vascularization after radiotherapy and due to the need to substitute larger defects after tumor excision. Aiming at introducing regenerative medicine for reconstruction after cancer, we tested an axially vascularized bone construct in an experimental setting that mimics the clinical situation after tumor resection and adjuvant radiotherapy. METHODS: Twenty bone constructs were axially vascularized using microsurgically created arteriovenous loops and were implanted subcutaneously in Lewis rats. After 2 weeks, the animals were randomly allocated either to receive a clinically relevant single dose of external beam irradiation or not (n = 10 for each group). The animals were sacrificed either after 1 week or 10 weeks after irradiation (n = 5 for each time point). The constructs were tested for vascularization, tissue growth, cellular proliferation, cellular apoptosis, and osteogenic differentiation via histomorphometric, immunohistochemical, and polymerase chain reaction (PCR) analysis. One construct per group was subjected at 10 weeks to qualitative micro-computed tomography (CT) imaging. RESULTS: Tissue generation and cellular proliferation were significantly reduced at 1 week after irradiation, but no longer significantly different after 10 weeks.No significant differences in vascularization were detected at any time point. Apoptosis did not show any statistically significant differences between both groups at both time points. At the late time point, mature bone was considerably more in the irradiated group, but the results were not statistically significant. PCR analysis showed a significantly enhanced expression of osteocalcin in the irradiated group at 1 week. Micro-CT imaging showed that both constructs were adequately vascularized with no evident morphologic differences regarding vascular density or vascular distribution. CONCLUSIONS: Axially vascularized bone constructs can withstand clinically relevant doses of irradiation and retain their angiogenic and osteogenic potential in the long term. Irradiation led to a delayed tissue generation with a comparatively enhanced osteogenic differentiation within the constructs.

Effect of blue LED on the healing process of third-degree skin burns: clinical and histological evaluation.

The aim of this study was to evaluate the effects of blue light-emitting diode (LED) on the healing process of third-degree skin burns in rats through clinical and histological parameters. Forty male Wistar rats were divided into two groups: control (CTR) (n = 20) and blue LED (BLUE) (n = 20), with subgroups (n = 5) for each time of euthanasia (7, 14, 21, and 28 days). LED (470 nm, 1 W, 12.5 J/cm2 per point, 28 s) was applied at four points of the wound (total, 50 J/cm2). Feed intake was measured every other day. It was observed that there were no statistically significant differences in the Wound Retention Index (WRI) of the BLUE group in relation to CTR group (p > 0.05) at the evaluation times. After 14, 21, and 28 days, it was observed that the animals in the BLUE group consumed more feed than animals in the CTR group (p < 0.05). At 7 days, there was a statistically significant increase in the angiogenic index (AI) in BLUE (median: 6.2) when compared to CTR (median: 2.4) (p = 0.01) and all animals in BLUE had already started re-epithelialization. This study suggests that blue LED, at the dosimetry used, positively contributed in important and initial stages of the healing process of third-degree skin burns.

Low level laser therapy (LLLT) modulates ovarian function in mature female mice.

It is known that LLLT has beneficial effects on several pathological conditions including wound healing, pain and inflammation. LLLT modulates biological processes, including cell proliferation, apoptosis and angiogenesis. In the present study, we examined the effect of local application of LLLT on follicular dynamics, ovarian reserve, AMH expression, progesterone levels, apoptosis, angiogenesis, and reproductive outcome in adult mice. LLLT (200 J/cm2) increased the percentage of primary and preantral follicles, whilst decreasing the percentage of corpora lutea compared to control ovaries. LLLT-treated ovaries did not exhibit any changes regarding the number of primordial follicles. We observed a higher percentage of AMH-positive follicles (in early stages of development) in LLLT-treated ovaries compared to control ovaries. LLLT reduced the P4 concentration and the apoptosis in early antral follicles compared to control ones. LLLT caused a reduction in the endothelial cell area and an increase in the periendothelial cell area in the ovary. Additionally, LLLT was able to improve oocyte quality. Our findings suggest that local application of LLLT modulates follicular dynamics by regulating apoptosis and the vascular stability in mouse ovary. In conclusion, these data indicate that LLLT might become a novel and useful tool in the treatment of several pathologies, including female reproductive disorders.