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.

Opsin 3 and 4 mediate light-induced pulmonary vasorelaxation that is potentiated by G protein-coupled receptor kinase 2 inhibition.

We recently demonstrated that blue light induces vasorelaxation in the systemic mouse circulation, a phenomenon mediated by the nonvisual G protein-coupled receptor melanopsin (Opsin 4; Opn4). Here we tested the hypothesis that nonvisual opsins mediate photorelaxation in the pulmonary circulation. We discovered Opsin 3 (Opn3), Opn4, and G protein-coupled receptor kinase 2 (GRK2) in rat pulmonary arteries (PAs) and in pulmonary arterial smooth muscle cells (PASMCs), where the opsins interact directly with GRK2, as demonstrated with a proximity ligation assay. Light elicited an intensity-dependent relaxation of PAs preconstricted with phenylephrine (PE), with a maximum response between 400 and 460 nm (blue light). Wavelength-specific photorelaxation was attenuated in PAs from Opn4-/- mice and further reduced following shRNA-mediated knockdown of Opn3. Inhibition of GRK2 amplified the response and prevented physiological desensitization to repeated light exposure. Blue light also prevented PE-induced constriction in isolated PAs, decreased basal tone, ablated PE-induced single-cell contraction of PASMCs, and reversed PE-induced depolarization in PASMCs when GRK2 was inhibited. The photorelaxation response was modulated by soluble guanylyl cyclase but not by protein kinase G or nitric oxide. Most importantly, blue light induced significant vasorelaxation of PAs from rats with chronic pulmonary hypertension and effectively lowered pulmonary arterial pressure in isolated intact perfused rat lungs subjected to acute hypoxia. These findings show that functional Opn3 and Opn4 in PAs represent an endogenous "optogenetic system" that mediates photorelaxation in the pulmonary vasculature. Phototherapy in conjunction with GRK2 inhibition could therefore provide an alternative treatment strategy for pulmonary vasoconstrictive disorders.

[Magnetic-laser influence on the system of nitric oxide and contractile activity of smooth muscles of rat aorta under hypertension].

The study was conducted on three groups of rats: Group I included Wistar rats with normal blood pressure (first control group); group II - rats with genetically determined hypertension (second control group); group Ill - rats with genetically determined hypertension under the influence ofmagnetic-laser power (study group). For the low-intensively magnetic-laser influence (MLI) we have used device MIT-MT, Ukraine, which was designed for the treatment of low-frequency magnetic field using optical flow blue and red ranges of spectrum. The MLI duration was 15 minutes for the blue range, and 25 minutes for the red one. Biochemical studies included the determination of the activity of isoenzymes of NO-synthase: constitutive (cNOS) and inducible (iNOS), the content of free hemoglobin, stable metabolites of NO, namely nitrite - (NO2(-)) and nitrate - (NO3(-)) anions, resistance to acid hemolysis of red blood cells. The contractile activity of smooth muscles of the aorta was measured. We found that magnetic-laser exposure of rats with genetically determined hypertension in the red (630 nm) and blue (470 nm wavelength) optical range even after a single session leads to an increased synthesis of nitric oxide in the blood plasma. Our data sindicate that the most effective in the intensification of endogenous nitric oxide (increase of NO2(-) and reduction of NO3(-)) and endothelium-dependent responses of aorta in rats with genetically determined hypertension was a ten-day course of the magnetic-laser exposure in the optical flow of the blue spectral range. Also, after 10 sessions of magnetic-laser exposure in rats from the above specified spectrum a stabilization of erythrocyte membranes was observed.

The impact of shock wave therapy at varied energy and dose levels on functional and structural changes in erectile tissue.

OBJECTIVES: Only minimal literature exists on consequences of shock wave therapy (SWT) on erectile function in treatment of Peyronie's disease (PD). This study was undertaken to define SWT impact at varied energy/dose levels at different time points on functional and structural changes in erectile tissue. METHODS: In 45 rats 2000 shock waves (sw) at 2 BAR were applied to the penis weekly sorted by one, two, and three sessions (high-dose/energy level, HD-1, HD-2, HD-3). Each group was followed for 1, 7, or 28 d before measuring intracavernosal pressure (ICP) and mean arterial pressure (MAP). Fifteen control animals (C1, C7, C28) underwent anesthesia alone. Another 15 animals were exposed to three SWT sessions applying 1000 sw at 1 BAR and analyzed identically (low-dose/energy level, LD-3-1, -7, -28). Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling assay was used to define the apoptotic index (AI) and Masson's trichrome (MT) staining was prepared to evaluate smooth muscle-to-collagen ratios. RESULTS: ICP/MAP ratios for all C groups displayed a mean of 64%. All SWT groups demonstrated significantly reduced ICP/MAP ratios compared to their corresponding C groups (p<0.05). The LD-3 groups showed a trend toward improved ICP/MAP ratios. LD-3-28 demonstrated significant recovery compared to HD-3-28 (55+/-8% vs. 41+/-10%, p=0.004), but remained reduced compared to C28 (63+/-5%, p=0.03). No statistical differences were seen for MT staining in SWT groups compared to C (p>0.05). AIs for the LD-3 groups were significantly lower compared to the HD-3 groups (p<0.001), but all AIs were significantly increased compared to C groups (p<0.01). CONCLUSIONS: Overall, at both energy/dose levels, SWT resulted in a time- and treatment-dependent reduction of ICP/MAP ratios, which might be mediated partly through apoptosis and collagenization of corporal smooth muscle.

Acute exposure to a moderate strength static magnetic field reduces edema formation in rats.

External application of static magnetic fields (SMF), used specifically for the treatment of inflammatory conditions such as soft tissue injuries, has recently become popular as a complementary and/or alternative therapy with minimal investigation into efficacy or mechanism. Localized inflammation was induced via injection of inflammatory agents lambda-carrageenan (CA) or histamine into rat hindpaws, alone or in conjunction with pharmacological agents, resulting in a spatially and temporally defined inflammatory reaction. Application of a 10- or 70-mT, but not a 400-mT, SMF for 15 or 30 min immediately following histamine-induced edema resulted in a significant, 20-50% reduction in edema formation. In addition, a 2-h, 70-mT field application to CA-induced edema also resulted in significant (33-37%) edema reduction. Field application before injection or at the time of maximal edema did not influence edema formation or resolution, respectively. Together, these results suggest the existence of a therapeutic threshold of SMF strength (below 400 mT) and a temporal dependence of efficacy. Administration of pharmacological agents directed at nitric oxide signaling and L-type Ca(2+) channel dynamics in conjunction with SMF treatment and histamine-induced edema revealed that the potential mechanism of SMF action may be via modulation of vascular tone through effects on L-type Ca(2+) channels in vascular smooth muscle cells.

Low-level laser irradiation modulates matrix metalloproteinase activity and gene expression in porcine aortic smooth muscle cells.

BACKGROUND AND OBJECTIVES: The vascular extracellular matrix is maintained by a dynamic balance between matrix synthesis and degradation. This equilibrium is disrupted in arterial pathologies such as abdominal aortic aneurysm. Low-level laser irradiation (LLLI) promotes wound healing. However, its effect on smooth muscle cells (SMCs), a central player in these responses, has not been established. The current study was designed to determine the effects of LLLI on arterial SMC proliferation, inflammatory markers, and matrix proteins. STUDY DESIGN/MATERIALS AND METHODS: Porcine primary aortic SMCs were irradiated with a 780 nm laser diode (1 and 2 J/cm(2)). Trypan blue exclusion assay, immunofluorescent staining for collagen I and III, Sircol assay, gelatin zymography, and RT-PCR were used to monitor proliferation; collagen trihelix formation; collagen synthesis; matrix metalloproteinase-2 (MMP-2) activity, and gene expression of MMP-1, MMP-2, tissue inhibitor of MMP-1 (TIMP-1), TIMP-2, and IL-1-beta, respectively. RESULTS: LLLI-increased SMC proliferation by 16 and 22% (1 and 2 J/cm(2), respectively) compared to non-irradiated cells (P<0.01 and P<0.0005). Immediately after LLLI, trihelices of collagen I and III appeared as perinuclear fluorescent rings. Collagen synthesis was increased twofold (2 days after LLLI: 14.3+/-3.5 microg, non-irradiated control: 6.6+/-0.7 microg, and TGF-beta stimulated control: 7.1+/-1.2 microg, P<0.05), MMP-2 activity after LLLI was augmented (over non-irradiated control) by 66+/-18% (2 J/cm(2); P<0.05), and MMP-1 gene expression upregulated. However, TIMP-2 was upregulated, and MMP-2 gene expression downregulated. IL-1-beta gene expression was reduced. CONCLUSIONS: LLLI stimulates SMC proliferation, stimulates collagen synthesis, modulates the equilibrium between regulatory matrix remodeling enzymes, and inhibits pro-inflammatory IL-1-beta gene expression. These findings may be of therapeutic relevance for arterial diseases such as aneurysm where SMC depletion, weakened extracellular matrix, and an increase in pro-inflammatory markers are major pathologic components.

Gamma irradiation induces acetylcholine-evoked, endothelium-independent relaxation and activates K-channels of isolated pulmonary artery of rats.

PURPOSE: To test the effects of irradiation (R*) on the pulmonary artery (PA). METHODS AND MATERIALS: Isolated PA rings were submitted to gamma irradiation (cesium, 8 Gy/min(-1)) at doses of 20 Gy-140 Gy. Rings were placed in an organ chamber, contracted with serotonin (10(-4) M 5-hydroxytryptamine [5-HT]), then exposed to acetylcholine (ACh) in incremental concentrations. Smooth muscle cell (SMC) membrane potential was measured with microelectrodes. RESULTS: A high dose of irradiation (60 Gy) increased 5HT contraction by 20%, whereas lower (20 Gy) doses slightly decreased it compared with control. In the absence of the endothelium, 5-HT precontracted rings exposed to 20 Gy irradiation developed a dose-dependent relaxation induced by acetylcholine (EI-ACh) with maximal relaxation of 60 +/- 17% (n = 13). This was totally blocked by L-NAME (10(-4) M), partly by 7-nitro indazole; it was abolished by hypoxia and iberiotoxin, decreased by tetra-ethyl-ammonium, and not affected by free radical scavengers. In irradiated rings, hypoxia induced a slight contraction which was never observed in control rings. No differences in SMC membrane potential were observed between irradiated and nonirradiated PA rings. CONCLUSION: Irradiation mediates endothelium independent relaxation by a mechanism involving the nitric oxide pathway and K-channels.

Cobalt-60 gamma radiation increased the nitric oxide generation in cultured rat vascular smooth muscle cells.

Radiation is a promising and new treatment for restenosis following angioplasty. Nitric oxide has been proposed as a potential "anti-restenotic" molecule. We radiated the cultured rat vascular smooth muscle cells with Cobalt-60 gamma radiation at doses of 14 and 25Gy and observed nitrite production, cGMP content, L-arginine uptake, inducible nitric oxide synthase (iNOS) activity, and the gene expression of iNOS. Results showed that radiation at doses of 14 and 25Gy increased cGMP content by 92.4% and 86.4%, respectively. Radiation at the dose of 25Gy increased the iNOS activity and nitrite content, but radiation at the dose of 14Gy had no significant effect on iNOS activity and NO production. Both doses of radiation significantly decreased the L-arginine transport. Radiation at the doses of 14 and 25Gy increased iNOS gene expression significantly, which was consistent with the effect of radiation on iNOS activity. In conclusion, radiation induces the NO generation by up-regulating the iNOS activity.

Canine carotid allograft changes after photochemotherapy and external polyester sheathing.

BACKGROUND: The purpose of this study is to define and refine the changes in canine carotid allografts after photochemotherapy and polyester sheathing. Photochemotherapy with 8-methoxy psoralen (8MOP) and UVA (PUVA) was given alone or combined with intraluminal visible light (VL) 450 nm in proper dosages to speed the depopulation of endothelial (EC) and smooth muscle cells (SMC) and to modulate the immune response. METHODS: Novel apparati were made for photochemotherapy of 19 right canine carotid arterial allografts with 19 paired untreated controls in the left carotid. External UVA and internal visible light (VL 450 nm) were used with 8-methoxy psoralen (8-MOP) as a sensitizer. RESULTS: With moderate dosage of 8-MOP (1 microg/cc) and 2-4 J/cm2 of external UVA (PUVA), smooth muscle cells (SMC) disappeared faster from the media and fibroblasts (FB) appeared earlier in the adventitia of the treated right allografts, reducing but not eliminating the immune response. Intraluminal VL did not enhance the PUVA effect. At 68 days, treated and control allografts showed similar dimensions with subsided immune reactions. The media thickness was reduced from 0.38 mm to 0.18 mm and the host adventitia increased from 0.22 to 0.60 mm. Variable reactions peaked between two and three weeks and subsided after one month. All allografts remained open with canine carotid i.d.s of 2-3 mm and 80 to 100 cc/ minute arterial flows. Although the UVA dosage was moderate) similar doses sterilized log 7 of staph aureus cultures in saline. The allografts without smooth muscle showed moderate but stable cylindrical dilatation without spasm or stenosis and with an adequate adventitial buttress for a small vessel. A polyester sleeve around four treated grafts was inseparable from the allograft in less than 3 weeks and tolerated well over a 70-day period. CONCLUSIONS: Biodegradable graft sheaths with bioerodible hydrogels with growth factors (FGF) for local delivery may provide a faster and more complete matrix remodeling for a superior conduit in the future.

Effects of low-dose-rate beta-irradiation on vascular smooth muscle cells: comparison with high-dose-rate exposure.

PURPOSE: Radiation therapy is undergoing extensive preclinical and clinical testing as a new tool to reduce restenosis after vessel injury. To date, however, no definite dose threshold has been identified after radioactive stent implantation. In this study, we compared the in vitro response of pig vascular smooth muscle cells (SMC) to conventional high-dose-rate (HDR) irradiation with the response to continuous low-dose rate (LDR) that could result from exposure to a radioactive stent. MATERIALS AND METHODS: Catheter-based radiotherapy delivers single doses at HDR whereas radioactive stents use a continuous LDR approach. Single doses in excess of 10 Gy have clearly shown a reduction in neointima formation and negative vessel remodeling in several animal models. Because dose rate is an important parameter modulating the overall biological response to ionizing radiation, we have compared the in vitro response of pig aortic SMC at conventional HDR (1.5 Gy/min) and at LDR (0.675 Gy/h). RESULTS: SMC showed significant repair of sublethal DNA damage and about twice the dose was necessary at the LDR to produce the same effect as that seen at the HDR. CONCLUSION: In vitro SMC exhibit a significant dose-rate effect that indicates that radioactive stents could deliver the dose at a sufficiently high dose rate to compensate for cell proliferation while at the same time the total dose should be increased to account for sublethal damage repair. This finding has important implications for the design of a radioactive stent.

Effects of ultraviolet light in vascular cells in vitro and in intact atherosclerotic explants: potential role of apoptosis in vascular biology.

Complex cell-to-cell interactions are known to participate during vascular injury and remodeling, resulting in smooth muscle cell proliferation. Mechanical interventions have yielded little benefit in limiting this process and several site-specific genetic therapies are not yet clinically available. The aim of this study was to delineate the effect of very short wavelength ultraviolet (UVC) light therapy on the viability of macrophage and smooth muscle cells. Vascular cells were both treated in vitro and in intact explanted atherosclerotic aortic segments ex vivo with UVC light. Brief exposure to short wavelength UVC light in the absence of photosensitizers elicited a differential temporal and functional response among treated cells. However, dramatic reduction in both cellular viability and proliferative capacity with eventual cell demise was observed in all UVC-treated cells. Flow cytometry and immunohistochemical analyses revealed the presence of extensive DNA fragmentation, suggestive of apoptosis as a predominant pathway of cell death in these cells exposed to UVC light. We hypothesize that selective induction of apoptosis, in contrast to necrosis, with UVC light may represent a beneficial approach to interdict the complex biologic cascade of messengers that participate in the restenotic response to vascular injury.

Effects of low-dose gamma-irradiation to the chest regions on the blood pressure of spontaneous hypertensive rats.

We examined changes in the blood pressure and the superoxide dismutase (SOD) activities in aortic tissue after gamma-irradiation to the chest regions of spontaneous hypertensive rats (SHR) and Wistar Kyoto rats (WKY), and obtained the following results. After 5 Gy irradiation, there was no change with time in the blood pressure in WKY rats, while a transient decrease (3 days later) was observed in SHR rats. The Cu/Zn-SOD activity in the aorta of SHR was less than that of WKY. 5 Gy irradiation induced an increase in the Cu/Zn-SOD activity in SHR nearly to the level observed in WKY, which remained unchanged upon irradiation. Therefore, unlike high-dose irradiation, irradiation at a relatively low-dose of 5 Gy to SHR appears to increase the aortic Cu/Zn-SOD activity, which is lacking in SHR, leading to a decrease in the blood pressure.