Blue laser-induced selective vasorelaxation by the activation of NOSs.

Phototherapy has been tried for treating cardiovascular diseases. In particular, ultraviolet and blue visible lights were suggested to be useful due to their nitric oxide (NO)-production ability in the skin. However, the effects of blue light on the arterial contractility are controversial. Here, we hypothesized that appropriate protocol of blue laser can induce selective vasorelaxation by activating vasodilating signaling molecules in arteries. Using organ chamber arterial mechanics, NO assay, Matrigel assay, and microarray, we showed that a 200-Hz, 300-µs, 445-nm pulsed-laser (total energy of 600 mJ; spot size 4 mm) induced selective vasorelaxation, without vasocontraction in rat mesenteric arteries. The laser stimulation increased NO production in the cord blood-endothelial progenitor cells (CB-EPCs). Both the laser-induced vasorelaxation and NO production were inhibited by a non-selective, pan-NO synthase inhibitor, L-NG-Nitro arginine methyl ester. Microarray study in CB-EPCs suggested up-regulation of cryptochrome (CRY)2 as well as NO synthase (NOS)1 and NOSTRIN (NOS trafficking) by the laser. In conclusion, this study suggests that the 445-nm blue puled-laser can induce vasorelaxation possibly via the CRY photoreceptors and NOSs activation. The blue laser-therapy would be useful for treating systemic hypertension as well as improving local blood flow depending on the area of irradiation.

In-vivo quantification of the revascularization of a human acellular dermis seeded with EPCs and MSCs in co-culture with fibroblasts and pericytes in the dorsal chamber model in pre-irradiated tissue.

INTRODUCTION: Transplantation of a cell-seeded graft may improve wound healing after radiotherapy. However, the survival of the seeded cells depends on a rapid vascularization of the graft. Co-culturing of adult stem cells may be a promising strategy to accelerate the vessel formation inside the graft. Thus, we compared the in vivo angiogenic potency of mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) using dorsal skinfold chambers and intravital microscopy. MATERIALS AND METHODS: Cells were isolated from rat bone marrow and adipose tissue and characterized by immunostaining and flow cytometry. Forty-eight rats received a dorsal skinfold chamber and were divided into 2 main groups, irradiated and non-irradiated. Each of these 2 groups were further subdivided into 4 groups: unseeded matrices, matrices + fibroblasts + pericytes, matrices + fibroblasts + pericytes + MSCs and matrices + fibroblasts + pericytes + EPCs. Vessel densities were quantified semi-automatically using FIJI. RESULTS: Fibroblasts + pericytes - seeded matrices showed a significantly higher vascular density in all groups with an exception of non-irradiated rats at day 12 compared to unseeded matrices. Co-seeding of MSCs increased vessel densities in both, irradiated and non-irradiated groups. Co-seeding with EPCs did not result in an increase of vascularization in none of the groups. DISCUSSION: We demonstrated that the pre-radiation treatment led to a significant decreased vascularization of the implanted grafts. The augmentation of the matrices with fibroblasts and pericytes in co-culture increased the vascularization compared to the non-seeded matrices. A further significant enhancement of vessel ingrowth into the matrices could be achieved by the co-seeding with MSCs in both, irradiated and non-irradiated groups.

TNF-TNFR2/p75 signaling inhibits early and increases delayed nontargeted effects in bone marrow-derived endothelial progenitor cells.

TNF-α, a pro-inflammatory cytokine, is highly expressed after being irradiated (IR) and is implicated in mediating radiobiological bystander responses (RBRs). Little is known about specific TNF receptors in regulating TNF-induced RBR in bone marrow-derived endothelial progenitor cells (BM-EPCs). Full body γ-IR WT BM-EPCs showed a biphasic response: slow decay of p-H2AX foci during the initial 24 h and increase between 24 h and 7 days post-IR, indicating a significant RBR in BM-EPCs in vivo. Individual TNF receptor (TNFR) signaling in RBR was evaluated in BM-EPCs from WT, TNFR1/p55KO, and TNFR2/p75KO mice, in vitro. Compared with WT, early RBR (1-5 h) were inhibited in p55KO and p75KO EPCs, whereas delayed RBR (3-5 days) were amplified in p55KO EPCs, suggesting a possible role for TNFR2/p75 signaling in delayed RBR. Neutralizing TNF in γ-IR conditioned media (CM) of WT and p55KO BM-EPCs largely abolished RBR in both cell types. ELISA protein profiling of WT and p55KO EPC γ-IR-CM over 5 days showed significant increases in several pro-inflammatory cytokines, including TNF-α, IL-1α (Interleukin-1 alpha), RANTES (regulated on activation, normal T cell expressed and secreted), and MCP-1. In vitro treatments with murine recombinant (rm) TNF-α and rmIL-1α, but not rmMCP-1 or rmRANTES, increased the formation of p-H2AX foci in nonirradiated p55KO EPCs. We conclude that TNF-TNFR2 signaling may induce RBR in naïve BM-EPCs and that blocking TNF-TNFR2 signaling may prevent delayed RBR in BM-EPCs, conceivably, in bone marrow milieu in general.

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.

Dickkopf-1 Treatment Stimulates Hematopoietic Regenerative Function in Infused Endothelial Progenitor Cells.

Acute high-dose radiation injury damages the bone marrow hematopoietic stem and progenitor cell compartment. This damage compromises the functional ability of the bone marrow to produce mature blood cells and results in an increased risk of death due to hematopoietic complications. Past work has shown that the bone marrow endothelium provides critical cues, which promote hematopoietic stem cell regeneration after injury. Additionally, transfusion of endothelial cells after radiation injury has been shown to promote recovery of both the bone marrow vasculature and hematopoietic systems. In this work, we examined the regenerative capacity of intravenous infusion of umbilical cord-blood derived endothelial progenitor cells (EPCs) since this is a cell source which is easy to obtain, expand and cryopreserve. We show that pre-treatment with the Wnt-antagonist Dickkopf1 (Dkk1) augments EPC regenerative function in an allogeneic mouse transplant model. Here, hematopoietic recovery was assessed in Balb/c mice after 5 Gy total-body irradiation and transplantation with C57/BL6-derived EPCs either with or without Dkk1 pre-treatment. The Dkk1-treated EPC group had significantly faster recovery of peripheral white blood cells, total bone marrow cellularity, bone marrow progenitors and BM endothelial cells compared to EPC treatment alone or saline controls. Importantly, after an LD50/30 dose of 8 Gy in the Balb/c mouse, Dkk1-treated EPCs were able to rescue 100% of irradiated mice versus 80% in the EPC control group and only 33% in the saline-treated group. To understand how Dkk1 induces regenerative function in the EPCs, we screened for pro-regenerative factors secreted by the EPC in response to Dkk1. Dkk1-treated EPCs were observed to secrete high levels of the anti-fibrotic protein follistatin as well as several proteins known to promote regeneration including EGF, VEGF and G-CSF. This work demonstrates the potential for Dkk1-treated EPCs as a rescue therapeutic for victims of acute radiation injury.

Effectiveness of endothelial progenitor cell culture under microgravity for improved angiogenic potential.

Endothelial progenitor cell (EPC) transplantation is beneficial for ischemic diseases such as critical limb ischemia and ischemic heart disease. The scarcity of functional EPCs in adults is a limiting factor for EPC transplantation therapy. The quality and quantity culture (QQc) system is an effective ex vivo method for enhancing the number and angiogenic potential of EPCs. Further, microgravity environments have been shown to enhance the functional potential of stem cells. We therefore hypothesized that cells cultured with QQc under microgravity may have enhanced functionality. We cultured human peripheral blood mononuclear cells using QQc under normal (E), microgravity (MG), or microgravity followed by normal (ME) conditions and found that ME resulted in the most significant increase in CD34+ and double positive Dil-Ac-LDL-FITC-Ulex-Lectin cells, both EPC markers. Furthermore, angiogenic potential was determined by an EPC-colony forming assay. While numbers of primitive EPC-colony forming units (pEPC-CFU) did not change, numbers of definitive EPC-CFU colonies increased most under ME conditions. Gene-expression profiling also identified increases in angiogenic factors, including vascular endothelial growth factor, under MG and ME conditions. Thus, QQc along with ME conditions could be an efficient system for significantly enhancing the number and angiogenic potential of EPCs.

Low-dose ionizing radiation induces therapeutic neovascularization in a pre-clinical model of hindlimb ischemia.

AIMS: We have previously shown that low-dose ionizing radiation (LDIR) induces angiogenesis but there is no evidence that it induces neovascularization in the setting of peripheral arterial disease. Here, we investigated the use of LDIR as an innovative and non-invasive strategy to stimulate therapeutic neovascularization using a model of experimentally induced hindlimb ischemia (HLI). METHODS AND RESULTS: After surgical induction of unilateral HLI, both hindlimbs of female C57BL/6 mice were sham-irradiated or irradiated with four daily fractions of 0.3 Gy, in consecutive days and allowed to recover. We demonstrate that LDIR, significantly improved blood perfusion in the murine ischemic limb by stimulating neovascularization, as assessed by laser Doppler flow, capillary density, and collateral vessel formation. LDIR significantly increased the circulating levels of VEGF, PlGF, and G-CSF, as well as the number of circulating endothelial progenitor cells (EPCs) mediating their incorporation to ischemic muscles. These effects were dependent upon LDIR exposition on the ischemic niche (thigh and shank regions). In irradiated ischemic muscles, these effects were independent of the recruitment of monocytes and macrophages. Importantly, LDIR induced a durable and simultaneous up-regulation of a repertoire of pro-angiogenic factors and their receptors in endothelial cells (ECs), as evident in ECs isolated from the irradiated gastrocnemius muscles by laser capture microdissection. This specific mechanism was mediated via vascular endothelial growth factor (VEGF) receptor signaling, since VEGF receptor inhibition abrogated the LDIR-mediated gene up-regulation and impeded the increase in capillary density. Finally, the vasculature in an irradiated non-ischemic bed was not affected and after 52 week of LDIR exposure no differences in the incidence of morbidity and mortality were seen. CONCLUSIONS: These findings disclose an innovative, non-invasive strategy to induce therapeutic neovascularization in a mouse model of HLI, emerging as a novel approach in the treatment of critical limb ischemia patients.

Irradiated human endothelial progenitor cells induce bystander killing in human non-small cell lung and pancreatic cancer cells.

Purpose To investigate whether irradiated human endothelial progenitor cells (hEPC) could induce bystander killing in the A549 non-small cell lung cancer (NSCLC) cells and help explain the improved radiation-induced tumor cures observed in A549 tumor xenografts co-injected with hEPC. Materials and methods We investigated whether co-injection of CBM3 hEPC with A549 NSCLC cells would alter tumor xenograft growth rate or tumor cure after a single dose of 0 or 5 Gy of X-rays. We then utilized dual chamber Transwell dishes, to test whether medium from irradiated CBM3 and CBM4 hEPC would induce bystander cell killing in A549 cells, and as an additional control, in human pancreatic cancer MIA PaCa-2 cells. The CBM3 and CBM4 hEPC were plated into the upper Transwell chamber and the A549 or MIA PaCa-2 cells were plated in the lower Transwell chamber. The top inserts with the CBM3 or CBM4 hEPC cells were subsequently removed, irradiated, and then placed back into the Transwell dish for 3 h to allow for diffusion of any potential bystander factors from the irradiated hEPC in the upper chamber through the permeable membrane to the unirradiated cancer cells in the lower chamber. After the 3 h incubation, the cancer cells were re-plated for clonogenic survival. Results We found that co-injection of CBM3 hEPC with A549 NSCLC cells significantly increased the tumor growth rate compared to A549 cells alone, but paradoxically also increased A549 tumor cure after a single dose of 5 Gy of X-rays (p < 0.05). We hypothesized that irradiated hEPC may be inducing bystander killing in the A549 NSCLC cells in tumor xenografts, thus improving tumor cure. Bystander studies clearly showed that exposure to the medium from irradiated CBM3 and CBM4 hEPC induced significant bystander killing and decreased the surviving fraction of A549 and MIA PaCa-2 cells to 0.46 (46%) ± 0.22 and 0.74 ± 0.07 (74%) respectively (p < 0.005, p < 0.0001). In addition, antibody depletion studies demonstrated that the bystander killing induced in both A549 and MIA PaCa-2 cells was mediated by the cytokines TNF-α and TGF-ß (p < 0.05). Conclusions These data provide evidence that irradiated hEPC can induce strong bystander killing in A549 and MIA PaCa-2 human cancer cells and that this bystander killing is mediated by the cytokines TNF-α and TGF-ß.

mESC-based in vitro differentiation models to study vascular response and functionality following genotoxic insults.

Because of high exposure to systemic noxae, vascular endothelial cells (EC) have to ensure distinct damage defense and regenerative mechanisms to guarantee vascular health. For meaningful toxicological drug assessments employing embryonic stem cell (ESC)-based in vitro models, functional competence of differentiated progeny and detailed knowledge regarding damage defense mechanisms are essential. Here, mouse ESCs (mESC) were differentiated into functionally competent vascular cells (EC and smooth muscle cells [SMC]). mESC, EC, and SMC were comparatively analyzed regarding DNA repair and DNA damage response (DDR). Differentiation was accompanied by both congruent and unique alterations in repair and DDR characteristics. EC and SMC shared the downregulation of genes involved cell cycle regulation and repair of DNA double-strand breaks (DSBs) and mismatches, whereas genes associated with nucleotide excision repair (NER), apoptosis, and autophagy were upregulated when compared with mESC. Expression of genes involved in base excision repair (BER) was particularly low in SMC. IR-induced formation of DSBs, as detected by nuclear γH2AX foci formation, was most efficient in SMC, the repair of DSBs was fastest in EC. Together with substantial differences in IR-induced phosphorylation of p53, Chk1, and Kap1, the data demonstrate complex alterations in DDR capacity going along with the loss of pluripotency and gain of EC- and SMC-specific functions. Notably, IR exposure of early vascular progenitors did not impair differentiation into functionally competent EC and SMC. Summarizing, mESC-based vascular differentiation models are informative to study the impact of environmental stressors on differentiation and function of vascular cells.