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.

Irradiation Accelerates Plaque Formation and Cellular Senescence in Flow-Altered Carotid Arteries of Apolipoprotein E Knock-Out Mice.

Background Chronic inflammation through cellular senescence, known as the senescence-associated secretory phenotype, is a mechanism of various organ diseases, including atherosclerosis. Particularly, ionizing radiation (IR) contributes to cellular senescence by causing DNA damage. Although previous clinical studies have demonstrated that radiotherapy causes atherosclerosis as a long-term side effect, the detailed mechanism is unclear. This study was conducted to investigate the relationship between radiation-induced atherosclerosis and senescence-associated secretory phenotype in murine carotid arteries. Methods and Results Partial ligation of the left carotid artery branches in 9-week-old male apolipoprotein E-deficient mice was performed to induce atherosclerosis. The mice received total body irradiation at a dose of 6 Gy using gamma rays at 2 weeks post operation. We compared the samples collected 4 weeks after IR with unirradiated control samples. The IR and control groups presented pathologically progressive lesions in 90.9% and 72.3% of mice, respectively. Plaque volume, macrophage accumulation, and phenotype switching of vascular smooth muscle cells were advanced in the IR group. Irradiated samples showed increased persistent DNA damage response (53BP1 [p53 binding protein 1]), upregulated cyclin-dependent kinase inhibitors (p16INK4a and p21), and elevated inflammatory chemokines expression (monocyte chemotactic protein-1, keratinocyte-derived chemokine, and macrophage inflammatory protein 2). Conclusions IR promoted plaque growth in murine carotid arteries. Our findings support the possibility that senescence-associated secretory phenotype aggravates atherogenesis in irradiated artery. This mice model might contribute to mechanism elucidation of radiation-induced atherosclerosis.

Irradiation abolishes smooth muscle investment into vascular lesions in specific vascular beds.

The long-term adverse effects of radiotherapy on cardiovascular disease are well documented. However, the underlying mechanisms responsible for this increased risk are poorly understood. Previous studies using rigorous smooth muscle cell (SMC) lineage tracing have shown abundant SMC investment into atherosclerotic lesions, where SMCs contribute to the formation of a protective fibrous cap. Studies herein tested whether radiation impairs protective adaptive SMC responses during vascular disease. To do this, we exposed SMC lineage tracing (Myh11-ERT2Cre YFP+) mice to lethal radiation (1,200 cGy) followed by bone marrow transplantation prior to atherosclerosis development or vessel injury. Surprisingly, following irradiation, we observed a complete loss of SMC investment in 100% of brachiocephalic artery (BCA), carotid artery, and aortic arch lesions. Importantly, this was associated with a decrease in multiple indices of atherosclerotic lesion stability within the BCA. Interestingly, we observed anatomic heterogeneity, as SMCs accumulated normally into lesions of the aortic root and abdominal aorta, suggesting that SMC sensitivity to lethal irradiation occurs in blood vessels of neural crest origin. Taken together, these results reveal an undefined and unintended variable in previous studies using lethal irradiation and may help explain why patients exposed to radiation have increased risk for cardiovascular disease.

Radiation suppresses neointimal hyperplasia through affecting proliferation and apoptosis of vascular smooth muscle cells.

PURPOSE: To study the effect of x-ray radiotherapy on vascular smooth muscle cells (VSMCs) and elucidate the mechanisms in preventing neointimal hyperplasia of prosthetic vascular grafts. MATERIALS AND METHODS: In model I, twelve mongrel dogs underwent revascularization with prosthetic grafts and half the dogs underwent irradiation of the grafts at 28 Gy. In model II, human VSMCs (hVSMCs) were maintained and divided into six groups to which external radiation was applied at six different doses: 0 Gy, 2 Gy, 8 Gy, 16 Gy, 24 Gy and 30 Gy. In both models, specimens were harvested and examined by using morphological, immunological, cellular and molecular methods. RESULTS: After irradiation, the neointima thickness was significantly lower in irradiated groups (p≤0.01). The radiotherapy could up-regulate p27kip1, and down-regulate proliferating cell nuclear antigen (PCNA) and S phase kinase associated protein 2 (Skp2). X-ray irradiation inhibits the proliferation of hVSMCs via acting on G1/S phase of cell cycle. The apoptosis of hVSMCs increased significantly with dose and time. The expression of PCNA and Skp2 were decreased after a first increasing trend with dose, but had a significant negative correlation with time. The expression of p27kip1 had a significant positive correlation with dose and time. CONCLUSIONS: Postoperative external fractionated irradiation after prosthetic vessel replacement of the abdominal aorta suppressed the development of hyperplasia in the graft neointima in the short term. There was a prominent time- and dose-dependent inhibition of VSMC proliferation by radiation when it was administered.

Low-power laser irradiation inhibits PDGF-BB-induced migration and proliferation via apoptotic cell death in vascular smooth muscle cells.

Vascular restenosis after injury of blood vessel has been implicated in various responses including apoptosis, migration, and proliferation in vascular smooth muscle cells (VSMCs) stimulated by diverse growth factors underlying platelet-derived growth factor (PDGF). Previous studies evaluated the effects of low-power laser (LPL) irradiation over various wavelength ranges on VSMC events in normal and pathologic states. However, whether VSMC responses are affected by LPL irradiation remains unclear. The purpose of this study is to explore the effects of LPL (green diode laser 532-nm pulsed wave of 300 mW at a spot diameter of 1 mm) irradiation on the responses, apoptosis, migration, and proliferation of VSMCs. The effect of LPL irradiation was tested on VSMCs through cytotoxicity, proliferation, migration, and apoptotic assays. Aortic ring assay was used to assess the effect of LPL irradiation on aortic sprout outgrowth. Protein expression levels were determined by western blotting. LPL irradiation did not affect VSMC viability but slightly attenuated PDGF-BB-induced proliferation in VSMCs. In addition, LPL irradiation inhibited PDGF-BB-evoked migration of VSMCs. Aortic sprout outgrowth in response to PDGF-BB was diminished in cells treated with LPL. In contrast, LPL irradiation evoked apoptosis in VSMCs in the presence of PDGF-BB. Similarly, activation of caspase-3 and Bax, as well as p38 mitogen-activated protein kinase (MAPK), in VSMCs treated with PDGF-BB was enhanced by exposure to LPL. These findings indicate that LPL irradiation induces vascular apoptosis via p38 MAPK activation and simultaneously inhibits VSMC proliferation and migration in response to PDGF-BB.

Human adipose-derived stem cell spheroid treated with photobiomodulation irradiation accelerates tissue regeneration in mouse model of skin flap ischemia.

Skin flap grafting is a form of transplantation widely used in plastic surgery. However, ischemia/reperfusion injury is the main factor which reduces the survival rate of flaps following grafting. We investigated whether photobiomodulation (PBM) precondition prior to human adipose-derived stromal cell (hASC) spheroid (PBM-spheroid) transplantation improved skin tissue functional recovery by the stimulation of angiogenesis and tissue regeneration in skin flap of mice. The LED had an emission wavelength peaked at 660 ± 20 nm (6 J/cm2, 10 mW/cm2). The expression of angiogenic growth factors in PBM-spheroid hASCs was much greater than that of not-PBM-treated spheroid or monolayer-cultured hASCs. From immunochemical staining analysis, the hASCs of PBM-spheroid were CD31+, KDR+, and CD34+, whereas monolayer-cultured hASCs were negative for these markers. To evaluate the therapeutic effect of hASC PBM-spheroid in vivo, PBS, monolayer-cultured hASCs, and not-PBM-spheroid were transplanted into a skin flap model. The animals were observed for 14 days. The PBM-spheroid hASCs transplanted into the skin flap ischemia differentiated into endothelial cells and remained differentiated. Transplantation of PBM-spheroid hASCs into the skin flap ischemia significantly elevated the density of vascular formations through angiogenic factors released by the skin flap ischemia and enhanced tissue regeneration at the lesion site. Consistent with these results, the transplantation of PBM-spheroid hASCs significantly improved functional recovery compared with PBS, monolayer-cultured hASCs, and not-PBM-spheroid treatment. These findings suggest that transplantation of PBM-spheroid hASCs may be an effective stem cell therapy for the treatment of skin flap ischemia.

Magnetically-responsive, multifunctional drug delivery nanoparticles for elastic matrix regenerative repair.

Arresting or regressing growth of abdominal aortic aneurysms (AAAs), localized expansions of the abdominal aorta are contingent on inhibiting chronically overexpressed matrix metalloproteases (MMPs)-2 and -9 that disrupt elastic matrix within the aortic wall, concurrent with providing a stimulus to augmenting inherently poor auto-regeneration of these matrix structures. In a recent study we demonstrated that localized, controlled and sustained delivery of doxycycline (DOX; a tetracycline-based antibiotic) from poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs), enhances elastic matrix deposition and MMP-inhibition at a fraction of the therapeutically effective oral dose. The surface functionalization of these NPs with cationic amphiphiles, which enhances their arterial uptake, was also shown to have pro-matrix regenerative and anti-MMP effects independent of the DOX. Based on the hypothesis that the incorporation of superparamagnetic iron oxide NPs (SPIONs) within these PLGA NPs would enhance their targetability to the AAA site under an applied external magnetic field, we sought to evaluate the functional effects of NPs co-encapsulating DOX and SPIONs (DOX-SPION NPs) on elastic matrix regeneration and MMP synthesis/activity in vitro within aneurysmal smooth muscle cell (EaRASMC) cultures. The DOX-SPION NPs were mobile under an applied external magnetic field, while enhancing elastic matrix deposition 1.5-2-fold and significantly inhibiting MMP-2 synthesis and MMP-2 and -9 activities, compared to NP-untreated control cultures. These results illustrate that the multifunctional benefits of NPs are maintained following SPION co-incorporation. Additionally, preliminary studies carried out demonstrated enhanced targetability of SPION-loaded NPs within proteolytically-disrupted porcine carotid arteries ex vivo, under the influence of an applied external magnetic field. Thus, this dual-agent loaded NP system proffers a potential non-surgical option for treating small growing AAAs, via controlled and sustained drug release from multifunctional, targetable nanocarriers. STATEMENT OF SIGNIFICANCE: Proactive screening of high risk elderly patients now enables early detection of abdominal aortic aneurysms (AAAs). There are no established drug-based therapeutic alternatives to surgery for AAAs, which is unsuitable for many elderly patients, and none which can achieve restore disrupted and lost elastic matrix in the AAA wall, which is essential to achieve growth arrest or regression. We have developed a first generation design of polymer nanoparticles (NPs) for AAA tissue localized delivery of doxycycline, a modified tetracycline drug at low micromolar doses at which it provides both pro-elastogenic and anti-proteolytic benefits that can augment elastic matrix regenerative repair. The nanocarriers themselves are also uniquely chemically functionalized on their surface to also provide them pro-elastin-regenerative & anti-matrix degradative properties. To provide an active driving force for efficient uptake of intra-lumenally infused NPs to the AAA wall, in this work, we have rendered our polymer NPs mobile in an applied magnetic field via co-incorporation of super-paramagnetic iron oxide NPs. We demonstrate that such modifications significantly improve wall uptake of the NPs with no significant changes to their physical properties and regenerative benefits. Such NPs can potentially stimulate structural repair in the AAA wall following one time infusion to delay or prevent AAA growth to rupture. The therapy can provide a non-surgical treatment option for high risk AAA patients.

A link between vascular damage and cognitive deficits after whole-brain radiation therapy for cancer: A clue to other types of dementia?

Whole brain radiation therapy for the treatment of tumors can sometimes cause cognitive impairment. Memory deficits were noted in up to 50% of treated patients over a short period of several months. In addition, an increased rate of dementia in young patients has been noted over the longer term, i.e. years. A deficit in neurogenesis after irradiation has been postulated to be the main cause of cognitive decline in patients, but recent data on irradiation therapy for limited parts of the brain appear to indicate other possibilities. Irradiation can directly damage various types of cells other than neuronal stem cells. However, this paper will focus on injury to brain vasculature leading to cognitive decline since vessels represent a better therapeutic target for drug development than other cells in the brain because of the blood-brain barrier.

Ultrasound triggered image-guided drug delivery to inhibit vascular reconstruction via paclitaxel-loaded microbubbles.

Paclitaxel (PTX) has been recognized as a promising drug for intervention of vascular reconstructions. However, it is still difficult to achieve local drug delivery in a spatio-temporally controllable manner under real-time image guidance. Here, we introduce an ultrasound (US) triggered image-guided drug delivery approach to inhibit vascular reconstruction via paclitaxel (PTX)-loaded microbubbles (PLM) in a rabbit iliac balloon injury model. PLM was prepared through encapsulating PTX in the shell of lipid microbubbles via film hydration and mechanical vibration technique. Our results showed PLM could effectively deliver PTX when exposed to US irradiation and result in significantly lower viability of vascular smooth muscle cells. Ultrasonographic examinations revealed the US signals from PLM in the iliac artery were greatly increased after intravenous administration of PLM, making it possible to identify the restenosis regions of iliac artery. The in vivo anti-restenosis experiments with PLM and US greatly inhibited neointimal hyperplasia at the injured site, showing an increased lumen area and reduced the ratio of intima area and the media area (I/M ratio). No obvious functional damages to liver and kidney were observed for those animals. Our study provided a promising approach to realize US triggered image-guided PTX delivery for therapeutic applications against iliac restenosis.

Differentiation Potential of Adipose-Derived Stem Cells When Cocultured with Smooth Muscle Cells, and the Role of Low-Intensity Laser Irradiation.

OBJECTIVE: The aim of the study was to investigate the differentiation potential of adipose-derived stem cells (ADSCs) when cocultured with smooth muscle cells (SMCs), and to determine the role of low-intensity laser irradiation (LILI). BACKGROUND DATA: ADSCs isolated from adipose tissue are isolated with ease and in large amounts. SMCs constitute most parts of the intestinal, urinary, reproductive, and cardiovascular systems. LILI has been found to have positive effects on different cell types, including ADSCs. METHODS: The study used ADSCs (Stempro Adipose Derived Stem Cells-R7788-115) and SMCs (SKU-T-1 American Type Culture Collection HTB-114) cell lines. These cell lines were cocultured in a 1:1 ratio with and without growth factors and then exposed to LILI using 636 nm at 5 J/cm2. RESULTS: Cell viability and proliferation increased significantly in the cocultured groups that were exposed to LILI alone, as well as in combination with growth factors. Further, there was a significant decrease in the expression of stem cell markers with a concomitant increase in SMC markers. CONCLUSIONS: These results suggest that ADSCs have the ability to differentiate into SMCs when cocultured with SMCs, whereas LILI potentially augments the differentiation potential and need. This further highlights the significant role that LILI has to offer ADSC therapy in regenerative medicine.

The mitochondrial lncRNA ASncmtRNA-2 is induced in aging and replicative senescence in Endothelial Cells.

Age-associated cardiovascular diseases are at least partially ascribable to vascular cell senescence. Replicative senescence (RS) and stress-induced premature senescence (SIPS) are provoked respectively by endogenous (telomere erosion) and exogenous (H2O2, UV) stimuli resulting in cell cycle arrest in G1 and G2 phases. In both scenarios, mitochondria-derived ROS are important players in senescence initiation. We aimed to define whether a mtDNA-transcribed long-non-coding-RNA (lncRNA), ASncmtRNA-2, has a role in vascular aging and senescence. Aortas of old mice, characterized by increased senescence, showed an increment in ASncmtRNA-2 expression. In vitro analysis of Endothelial Cells (EC) and Vascular Smooth Muscle Cells (VSMC) established that ASncmtRNA-2 is induced in EC, but not in VSMC, during RS. Surprisingly, ASncmtRNA-2 is not upregulated in two different EC SIPS scenarios, treated with H2O2 and UV. The p16 gene displayed similar ASncmtRNA-2 expression patterns, suggesting a possible co-regulation of the two genes. Interestingly, the expression of two miRNAs, hsa-miR-4485 and hsa-miR-1973, with perfect homology to the double strand region of ASncmtRNA-2 and originating at least in part from a mitochondrial transcript, was induced in RS, opening to the possibility that this lncRNA functions as a non-canonical precursor of these miRNAs. Cell cycle analysis of EC transiently over-expressing ASncmtRNA-2 revealed an accumulation of EC in the G2/M phase, but not in the G1 phase. We propose that ASncmtRNA-2 in EC might be involved in the RS establishment by participating in the cell cycle arrest in G2/M phase, possibly through the production of hsa-miR-4485 and hsa-miR-1973. This article is part of a Special Issue entitled: Mitochondria.

Low-frequency ultrasound induces apoptosis of rat aortic smooth muscle cells (A7r5) via the intrinsic apoptotic pathway.

Ultrasound, a non-invasive therapy method, is a potential tool for medical applications, but its biological effects on vascular smooth muscle cells (VSMCs) have not been characterized. The aim of this study was to explore the effect and possible apoptotic mechanism of VSMCs that were induced by low-frequency ultrasound (LFU). Cell viability and apoptosis of A7r5 cells were evaluated after treating A7r5 cells with a continuous 45-kHz 1.0-W/cm(2) ultrasound (exposure time of 0, 10, 20, 30, and 35 s) by MTT assay and flow cytometry. At the optimum ultrasound exposure condition (30 s), gene chip analysis was performed, and the apoptotic signaling pathway was confirmed by reverse transcription-polymerase chain reaction and Western blot. As measured by flow cytometry, LFU significantly induced A7r5 cell apoptosis. Comparing the ultrasound group with the control group, the protein expression of caspase-9 and caspase-3 was increased by 50 and 57%, respectively; the caspase-3 mRNA level was increased by 37.5%. These findings indicate that an intrinsic pathway plays a major role in apoptosis that is induced by LFU and that LFU can induce A7r5 cell apoptosis via caspase-9- and caspase-3-dependent pathways.

γ-Rays-generated ROS induce apoptosis via mitochondrial and cell cycle alteration in smooth muscle cells.

PURPOSE: γ-rays (IR) cause an increase in intracellular calcium [Ca(2+)], alters contractility and triggers apoptosis via the activation of protein kinase C in intestinal guinea pig smooth muscle cells. The present study investigated the role of the mitochondria in these processes and characterized proteins involved in IR-induced apoptosis. MATERIALS AND METHODS: Intestinal smooth muscle cells were exposed to 10-50 Gy from a (60)Co γ-source. Reactive oxygen species (ROS) levels were measured by colourimetry with a fluorescente probe. Protein expression was analyzed by immunoblotting and immunofluorescence. RESULTS: Apoptosis was inhibited by glutathione, possible by inhibiting the generation or scavenging ROS. Apoptosis was mediated by the mitochondria releasing cytochrome c leading to caspase 3 activation. IR increased the expression of the cyclins A, B2 and E and led to unbalanced cellular growth in an absorption dose-dependent manner. However, radiation did not induce alterations in the mitochondrial ultrastructure or in transmembrane electric potential. In contrast, IR increased the nuclear expression of cytoplasmic proteins and cyclins A and E. CONCLUSION: Smooth muscle cells subjected to IR undergo mitochondrial-mediated apoptosis that involves oncoproteins activation and preserves mitochondrial structure. IR also cause alterations in the expression and localization of both pro- and anti-apoptotic proteins.

Radiation therapy for dialysis access stenosis: unfulfilled promise or false expectations.

Hemodialysis vascular access dysfunction is a major cause of morbidity and hospitalization in the hemodialysis population at a cost of well over $1 billion per annum. Venous stenosis (due to venous neointimal hyperplasia [VNH]) is the most common cause of polytetrafluroethylene PTFE) dialysis access graft and arteriovenous fistula (AVF) failure. Despite the magnitude of the clinical problem, however, there are currently no effective therapies for this condition. We and others have previously demonstrated that VNH in PTFE dialysis grafts and AVF is composed of smooth muscle cells/myofibroblasts, endothelial cells within neointimal microvessels, and peri-graft macrophages. Radiation therapy blocks the proliferation and activation of all these cell types. The current review will dissect out the available in vitro, experimental, and clinical data on the use of radiation therapy for vascular stenosis in general, and for dialysis access dysfunction in particular. It is important to try and identify whether there is still a role for radiation therapy in this specific clinical setting. We believe that this is a critically important question to answer in view of the huge unmet clinical need that is currently associated with hemodialysis vascular access dysfunction.

Volume-dependent expression of in-field and out-of-field effects in the proton-irradiated rat lung.

PURPOSE: To investigate whether occurrence of early radiation effects in lung tissue depends on local dose only. METHODS AND MATERIALS: Twenty-five percent, 50%, 66%, 88%, or 100% of the rat lung was irradiated using single fractions of 150-MeV protons. For all volumes, in-field and out-of-field dose-response curves were obtained 8 weeks after irradiation. The pathohistology of parenchymal inflammation, infiltrates, fibrosis, and vascular damage and the relative expression of proinflammatory cytokines interleukin (IL)-1α, transforming growth factor-ß, IL-6, and tumor necrosis factor-α were assessed. RESULTS: For all histologic endpoints, irradiated dose- and volume-dependent in-field and out-of-field effects were observed, albeit with different dynamics. Of note, the out-of-field effects for vascular damage were very similar to the in-field effects. Interestingly, only IL-6 showed a clear dose-dependent increase in expression both in-field and out-of-field, whereas the expression levels of IL-1α, transforming growth factor-ß, and tumor necrosis factor-α were either very low or without a clear dose-volume relation. As such, none of the radiation effects studied depended only on local dose to the tissue. CONCLUSION: The effects of radiation to lung tissue do not only depend on local dose to that tissue. Especially at high-volume irradiation, lung damage seems to present globally rather than locally. The accuracy of predictive modeling may be improved by including nonlocal effects.

A dual-inhibition study on vascular smooth muscle cells with meclofenamic acid and ß-irradiation for the prevention of restenosis.

PURPOSE: Restenosis is still one of the major limitations after angioplasty. A therapeutic treatment combining ß-irradiation and pharmacologic cyclooxygenase-2 inhibition was employed to study the impact on vascular smooth muscle cells (SMCs). MATERIALS AND METHODS: The effects of meclofenamic acid in combination with yttrium-90 ((90)Y) on cell growth, clonogenic activity, cell migration, and cell cycle distribution of human aortic SMCs were investigated. Treatment was sustained over a period of 4 days and recovery of cells was determined until day 20 after initiation. The hypothesis was that there is no difference between control and treated groups. RESULTS: A dose-dependent growth inhibition was observed in single and combined treatment groups for meclofenamic acid and ß-irradiation. Cumulative radiation dosage of 8 Gy completely inhibited colony formation. This was also observed for 200 µM meclofenamic acid alone or in combination with minor ß-irradiation dosages. Results of the migration tests showed also a dose dependency with additive effects of combined therapy. Meclofenamic acid 200 µM alone and with cumulative ß-irradiation dosages resulted in an increased G2/M-phase share. CONCLUSIONS: Incubating human SMCs with meclofenamic acid and (90)Y for a period of 4 d (ie, 1.5 half-life times) resulted in an effective inhibition of smooth muscle cell proliferation, colony formation, and migration.

Oxidized low-density lipoprotein increases osteopontin expression by generation of oxidative stress.

Osteopontin (OPN) is an important mediator of inflammation and is involved in the generation of atherosclerotic lesions. Oxidized LDL (OxLDL) increased the intracellular and secreted levels of OPN in rat smooth muscle cells in a dose- and time-dependent manner. Experiments with kinase inhibitors demonstrated that this effect was mediated by ERK and JNK, but not p38. OxLDL induced oxidative stress, measured by the intracellular levels of reactive oxygen species (ROS) and lipid peroxidation products. The increase in OPN levels was reproduced by the lipid extract of the particle and prevented by the antioxidant vitamin E. Furthermore, ROS generated by UVA irradiation or treatment with pro-oxidant compounds such as buthionine sulfoximine or H(2)O(2) also enhanced intracellular and secreted OPN. Finally, OxLDL also augmented OPN levels in other cell types such as fibroblasts, keratinocytes, and endothelial cells. This work demonstrates the role of OxLDL in the expression of the OPN gene and further highlights the role of oxidative stress in the regulation of this cytokine. This might be related to the proinflammatory effects of OxLDL in the initiation and progression of atherosclerotic plaque.

Angiotensin II type 1 receptor-associated protein prevents vascular smooth muscle cell senescence via inactivation of calcineurin/nuclear factor of activated T cells pathway.

Emerging new research suggests that the functions of the angiotensin (Ang) II type 1 (AT(1)) receptor are regulated in a complex manner. AT(1) receptor-associated protein (ATRAP) has been reported to reduce AT(1) receptor signaling with enhancement of AT(1) receptor internalization and to regulate the calcineurin/nuclear factor of activated T cells (NFAT) pathway. We examined the possibility that ATRAP could attenuate AT(1) receptor-mediated vascular senescence via inactivation with the calcineurin/NFAT pathway. Ang II stimulation significantly increased senescence-associated beta-galactosidase (SA-beta-gal)-stained cells, oxidative stress, and expression of p53 and p21 in wild-type (WT) vascular smooth muscle cells (VSMC). Moreover, in WT VSMC, Ang II stimulation enhanced NFAT transcriptional activity, which was prevented by CAML-siRNA treatment. NFAT-siRNA treatment attenuated Ang-II-increased SA-beta-gal activity and p53 and p21 expression. Treatment with a calcineurin activity inhibitor, cyclosporin A, reduced Ang-II-induced NFAT transcriptional activity and senescent VSMC. In contrast, VSMC prepared from ATRAP transgenic (ATRAP-Tg) mice exhibited attenuation of Ang-II-induced SA-beta-gal activity, oxidative stress, NFAT transcriptional activity, and expression of p53 and p21. Moreover, ATRAP-Tg VSMC showed a more reduction of Ang-II-induced NFAT transcriptional activity by CAML-siRNA treatment than WT VSMC. Furthermore, we demonstrated that in ATRAP-Tg VSMC, NFAT activity and senescent cells induced by ultraviolet irradiation were decreased compared with those in WT VSMC. Treatment with an AT(1) receptor blocker, valsartan, blocked these senescent cells but did not change NFAT activity in both cells. These results suggest that ATRAP negatively regulates VSMC senescence by reducing AT(1) receptor signaling, and that ATRAP-mediated inactivation of the calcineurin/NFAT pathway could be at least partly involved in prevention of VSMC senescence, irrespective of AT(1) receptor blockade in some conditions.

Inhibition of the cystathionine-gamma-lyase/hydrogen sulfide pathway in rat vascular smooth muscle cells by cobalt-60 gamma radiation.

BACKGROUND: Radiation is a promising treatment for in stent restenosis and restenosis following percutaneous transluminal coronary angioplasty, which has troubled interventional cardiologists for a long time. It inhibits neointima hyperplasia, vascular remodeling, and increases the mean luminal diameter. The mechanism of intracoronary brachytherapy for restenosis is not well understood. Endogenous gaseous transmitters including nitric oxide and carbon monoxide are closely related to restenosis. Hydrogen sulfide, a new endogenous gaseous transmitter, is able to inhibit the proliferation of vascular smooth muscle cells and vascular remodeling. This study aimed to clarify the effect of radiation on cystathionine-gamma-lyase/hydrogen sulfide pathway in rat smooth muscle cells. METHODS: We studied the effect of radiation on the cystathionine-gamma-lyase/hydrogen sulfide pathway. Rat vascular smooth muscle cells were radiated with (60)Co gamma at doses of 14 Gy and 25 Gy respectively. Then the mRNA level of cystathionine-gamma-lyase was studied by quantitative reverse-transcription competitive polymerase chain reaction. Hydrogen sulfide concentration in culture medium was determined by methylene blue spectrophotometry. Cystathionine-gamma-lyase activity in vascular smooth muscle cells was also studied. RESULTS: (60)Co gamma radiation at a dose of 1 Gy did not affect the cystathionine-gamma-lyase/hydrogen sulfide pathway significantly. However, (60)Co gamma radiation at doses of 14 Gy and 25 Gy decreased the hydrogen sulfide synthesis by 21.9% (P<0.05) and 26.8% (P<0.01) respectively. At the same time, they decreased the cystathionine-gamma-lyase activity by 15.1% (P<0.05) and 20.5% (P<0.01) respectively, and cystathionine-gamma-lyase mRNA expression by 29.3% (P<0.01) and 38.2% (P<0.01) respectively. CONCLUSION: Appropriate (60)Co gamma radiation inhibits the H(2)S synthesis by inhibiting the gene expression of cystathionine-gamma-lyase and the cystathionine-gamma-lyase activity.

Effect of low level laser therapy on bronchial hyper-responsiveness.

The objective of this study was to investigate whether low level laser therapy (LLLT) could reduce bronchial hyper-responsiveness (BHR) induced by tumour necrosis factor-alpha (TNF-alpha) modulating the metabolism of inositol phosphate (IP) in bronchial smooth muscle cells (BSMCs). The study was on 28 Wistar rats, randomly divided into four groups. Irradiation (1.3 J/cm(2)) was administered 5 min and 4 h after bronchial smooth muscle (BSM) had been suspended in TNF-alpha baths, and the contractile response-induced calcium ion (Ca(2+)) sensitization was measured. The BSMCs were isolated, and the IP accumulation was measured before and after TNF-alpha immersion in the groups that had been irradiated or not irradiated. BSM segments significantly increased contraction 24 h after TNF-alpha immersion when exposed to carbachol (CCh) as Ca(2+), but it was significantly reduced by 64% and 30%, respectively, after laser treatment. The increase in IP accumulation induced by CCh after TNF-alpha immersion was reduced in the BSMCs by LLLT. The dose of 2.6 J/cm(2) reduced BHR and IP accumulation in the rats' inflammatory BSMCs.