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Low-level laser irradiation inhibits abdominal aortic aneurysm progression in apolipoprotein E-deficient mice

Lilach Gavish, Chen Rubinstein, Atilla Bulut, Yacov Berlatzky, Ronen Beeri, Dan Gilon, Leah Gavish, Mickey Harlev, Petachia Reissman, S. David Gertz
DOI: http://dx.doi.org/10.1093/cvr/cvp149 785-792 First published online: 14 May 2009

Abstract

Aims Increased early detection of abdominal aortic aneurysm (AAA) and the severe complications of its current treatment have emphasized the need for alternative therapeutic strategies that target pathogenetic mechanisms of progression and rupture. Recent in vitro studies from our laboratory have shown that low-level laser irradiation (LLLI) (780 nm) modifies cellular processes fundamental to aneurysm progression. The present study was designed to determine whether LLLI retards the progression of suprarenal AAA in vivo.

Methods and results High-frequency ultrasonography (0.01 mm resolution) was used to quantify the effect of LLLI on aneurysmatic aortic dilatation from baseline to 4 weeks after subcutaneous infusion of angiotensin II by osmotic minipumps in the apolipoprotein E-deficient mouse. At 4 weeks, seven of 15 non-irradiated, but none of the 13 LLLI, mice had aneurysmal dilatation in the suprarenal aneurysm-prone segments that had progressed to ≥50% increase in maximal cross-sectional diameter (CSD) over baseline (P = 0.005 by Fisher's exact test). The mean CSD of the suprarenal segments (normalized individually to inter-renal control segments) was also significantly lower in irradiated animals (LLLI vs. non-irradiated: 1.32 ± 0.14 vs. 1.82 ± 0.39, P = 0.0002 by unpaired, two-tailed t-test) with a 94% reduction in CSD at 4 weeks compared with baseline. M-mode ultrasound data showed that reduced radial wall velocity seen in non-treated was significantly attenuated in the LLLI mice, suggesting a substantial effect on arterial wall elasticity.

Conclusion These in vivo studies, together with previous in vitro studies from this laboratory, appear to provide strong evidence in support of a role for LLLI in the attenuation of aneurysm progression. Further studies in large animals would appear to be the next step towards testing the applicability of this technology to the human interventional setting.

  • Low-level laser
  • Apolipoprotein E-deficient mice
  • Aneurysm
  • Angiotensin-II
  • High-frequency ultrasound

1. Introduction

Increased detection of abdominal aortic aneurysm (AAA) at early stages of the disease1 and the severe complications often associated with currently available surgical and endovascular repair24 have emphasized the need for alternative therapeutic strategies that target pathogenetic mechanisms of progression and rupture.5

Low-level (non-thermal) laser irradiation (LLLI) in the red and far red ranges of the visible light spectrum and the near infrared regions (600 and 1000 nm) has been used for over 40 years in a variety of clinical situations.68 These include chronic recalcitrant wounds in diabetics,9 Achilles tendonitis,10 radiation-induced dermatitis,11 carpal tunnel syndrome,12 rheumatoid arthritis,13 and microcrystalline-induced arthropathies.14,15

Over the past 8–10 years, there has been increasing interest in the effects of LLLI on components of the cardiovascular system in normal and pathologic states. These studies have included effects on arterial and venous endothelium,16,17 smooth muscle cells,18 cardiac muscle cells,19,20 fibroblasts,21,22 monocyte/macrophages,23 and the erythrocytes themselves.2426

In vitro studies in our laboratory have shown that LLLI (780 nm) modifies certain processes fundamental to aneurysm progression. We found that LLLI increases porcine aortic smooth muscle cell proliferation, increases extracellular matrix protein expression and secretion, modulates activity and expression of matrix metalloproteinases, and inhibits gene expression of the pro-inflammatory cytokine interleukin (IL)-1β from these cells.18

Inflammation is a major component of all arteriosclerotic diseases including aneurysm with macrophage recruitment and secretion of pro-inflammatory cytokines being central to most immune responses in the arterial wall. In studies of the effect of 780 nm low-power laser on lipopolysaccharide-activated RAW macrophages,23 we found marked inhibition of gene expression of monocyte chemotactic protein-1 (MCP-1) and the pro-inflammatory cytokines, IL-1α, IL-1β, and IL-6, but also inhibition of the suppressor cytokine IL-10. We also found that LLLI reduced the secretion of the proteins themselves with MCP-1 and IL-1β being the representative examples.

These ostensibly contradictory effects of LLLI—on the one hand, pro-proliferative and pro-atherosclerotic, but on the other hand, anti-inflammatory and anti-atherosclerotic—suggested that this modality might be of therapeutic value for arterial diseases such as aneurysm where inflammatory processes and weakening of the matrix structure of the arterial wall are major pathologic components. In the present study, we used high-frequency ultrasonography to determine whether LLLI might retard the progression of AAA in vivo.

2. Methods

2.1 Study design

For these studies, we used the angiotensin II-infused apolipoprotein E-deficient (−/−) C57/Black6 mouse model developed in the laboratory of Daugherty et al.27 In this mouse model, suprarenal AAAs form in up to 80–85% of the cases.2731 High-frequency ultrasonography (0.01 mm resolution) was used for quantification of the effect of LLLI on aortic expansion over time. This recently developed technology was designed specifically for non-invasive microimaging in mice.30

Angiotensin II was infused in 38 male mice aged 12–13 weeks via subcutaneously implanted osmotic minipumps (see details below). Laparotomy was performed to enable direct irradiation of the aorta. Nine animals died during surgery, and one was disqualified as a result of pump extrusion. Of the 28 mice, 13 were irradiated and 15 were sham-operated, non-irradiated controls.

Morphometric ultrasonographic measurements of the suprarenal aneurysm-prone segment and the adjacent inter-renal non-aneurysm-prone segments were performed at baseline and at 4 weeks after the onset of angiotensin II infusion.

2.2 Mice

The mice were bred in-house from stock originating from Jackson Laboratories. The mice were housed in a specific pathogen-free environment. Water and normal diet were available ad libitum. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Animal care and the experimental procedures were approved by the Ethics Committee of the Faculty of Medicine of The Hebrew University, Jerusalem, Israel (MD-07-10349-3).

2.3 Angiotensin II infusion

Osmotic minipumps (Alzet, model 2004, Durect Corp., Cupertino, CA, USA) were filled with angiotensin II (Calbiochem, La Jolla, CA, USA) (infusion rate 1000 ng/kg/min). The pumps were implanted subcutaneously on the right flank through an incision in the scapular region and maintained for the entire 28 days—from the time of irradiation until sacrifice.

2.4 Low-level laser irradiation

A diode laser system coupled to an optic fibre was used with 0–450 mW power and 780 nm wavelength (BWTek, Newark, DE, USA). The irradiation box contained two compartments with a hole between. The laser was placed in the upper compartment with the optic fibre tip threaded through the hole above the irradiation plane at a distance adjusted for optimal expansion of the ray. The power was measured at the plane of the aorta with a Laser Mate power meter (Coherent, Auburn group, Europe). The exposed aorta was irradiated at 4 mW/cm2 for 9 min which accumulated to a total energy density of 2 J/cm2.

2.5 Surgical protocol

Mice were anaesthetized by subcutaneous injection of ketamine (200 mg/kg) and xylazine (10 mg/kg). All animals received subcutaneous injections of cefamizine (30 mg/kg), tramadol analgesia (2 mg/kg), and warmed saline (2 mL). Chloramphenicol ointment was applied locally to the conjunctival sacs to prevent corneal damage. The abdominal aorta was exposed through a left subcostal incision (retroperitoneal approach32), and the region between the diaphragm and the renal arteries was isolated from the surrounding retroperitoneal structures. The mice were then placed in the irradiation box with the exposed abdominal aorta localized in the centre of the beam. The sham-operated, non-irradiated control animals followed the same protocol but with the laser turned off. The Alzet minipump was implanted as described above.

2.6 Ultrasound imaging and analysis

The high-resolution ultrasound imaging system Vevo 770, VisualSonics, Toronto, Canada, was used to perform two-dimensional (B-mode) and motion-mode (M-mode) imaging using a mechanical transducer (RMV707B) synchronized to the electrocardiographic signal. The transducer had a central frequency of 40 MHz, a focal length of 6 mm, a frame rate of 30 Hz, and an 8 × 8 mm field of view with spatial resolution of 30 µm. Scans were performed under anaesthesia using 2% isofluorane. A longitudinal image of the abdominal aorta between the diaphragm and the renal arteries was acquired. Doppler signals were used to confirm the identification of the abdominal aorta. Transverse images at the level of the maximal dilatation of the aneurysm-prone suprarenal portion of the abdominal aorta were acquired in B-mode and M-mode with the adjacent, non-aneurysm-prone portion of the aorta between the right and left renal arteries serving as internal controls (Figure 1). The measurements were performed with VisualSonics proprietary software using multiple frames. The maximum aortic cross-sectional diameter (CSD) (associated with systole) was determined from B-mode data. Diastolic diameter, systolic diameter, and maximal aortic radial wall velocity (RWV) [the first derivative (slope) of the aortic diameter with respect to time (dD/dt)] were determined from M-mode to assess the consistency and visco-elastic behaviour of the arterial wall.33 Pulse diameter was calculated by subtracting diastolic from systolic aortic diameter and then normalizing to maximum systolic diameter to account for vessel size.

Figure 1

Planes of ultrasound measurements: upper dotted line, site of maximal dilatation of the aneurysm-prone suprarenal abdominal aorta; lower dotted line, adjacent non-aneurysm-prone aortic segment between the right and left renal arteries.

For morphometric analysis, the number of individual mice with ≥50, ≥40, or ≥30% CSD expansion of the suprarenal aortic segments 28 days after baseline was determined for control and LLLI mice. In addition to the individual (categorical) data, the morphometric data were also analysed after calculating the mean CSD across all animals in each group (continuous data). In order to account for any normal variations in vessel sizes between animals, measurements of the suprarenal segments were also normalized to the adjacent, non-dilated, internal control segments of each animal at the level between the origins of the left and right renal arteries.

Categorical and continuous analyses were also conducted for the physiological parameters derived from M-mode data—pulse diameter (PD) normalized to systole and maximal RWV. The number of individual mice showing ≥75% reduction of PD and the number of mice showing ≥50% reduction in maximal RWV at endpoint over baseline were determined for control and LLLI mice. The means of these two parameters were calculated across all animals in each of the four groups.

2.7 Statistical analysis

Categorical data were analysed using Fisher's exact test with P < 0.05 considered significant. For continuous data, comparisons between measurements at baseline and at the 28-day endpoint in the same mice were performed by paired, two-tailed t-test with the Bonferroni correction for multiple comparisons. Comparisons between control and LLLI mice at baseline or at the 28-day endpoint were performed by unpaired, two-tailed t-test with the Bonferroni correction.

Data from the M-mode measurements of one animal were excluded because of technical problems with the 28-day endpoint measurement. Data of RWV from one additional animal were excluded on the basis of outlier analysis.34

3. Results

Ultrasound measurements for each animal are presented in Table 1.

View this table:
Table 1

Ultrasound measurements

Time pointBaselineEndpoint
Ultrasound modeB-modeM-modeB-modeM-mode
Group#MSR-BRen-BDias-BSyst-BVeloc-BMSR -ERen-EDias-ESyst-EVeloc-E
Control11.141.051.021.214.31.410.941.271.371.6
Control21.180.941.191.314.131.510.841.411.613.31
Control31.160.921.191.33.232.320.842.12.110.73
Control41.160.991.151.33.911.70.941.731.791.6
Control51.050.721.041.152.461.720.961.61.72.23
Control61.140.861.171.322.721.690.821.541.652.95
Control71.060.771.021.153.021.840.941.942.13.57
Control81.110.881.051.23.771.960.911.331.351.37
Control91.110.811.161.313.921.340.961.181.427.6a
Control101.170.791.051.193.641.811.05TPTPTP
Control111.080.760.961.114.672.3612.292.340.96
Control1210.831.021.174.921.581.051.291.54.52
Control131.220.881.091.273.51.431.051.161.343.92
Control141.250.881.151.262.121.640.981.361.52.12
Control151.140.871.161.254.571.531.021.671.71.53
LLLI161.040.90.931.033.91.280.951.031.216.28
LLLI171.050.881.021.112.991.280.911.11.34.8
LLLI181.150.91.021.183.351.610.941.531.62.19
LLLI191.170.961.111.282.051.050.780.871.043.14
LLLI201.230.921.091.214.471.571.151.511.572.89
LLLI211.040.781.041.231.261.041.071.253.72
LLLI2210.890.911.053.871.31.041.231.373.31
LLLI231.080.961.121.274.041.191.021.061.164.29
LLLI241.10.850.981.153.921.361.021.161.353.98
LLLI251.190.821.041.264.561.511.131.521.615.3
LLLI261.110.881.031.183.41.381.141.371.54.64
LLLI271.170.821.031.244.011.321.081.371.52.69
LLLI281.230.81.071.244.611.150.881.221.354.56
  • LLLI, low-level laser irradiation; MSR, maximal suprarenal diameter (mm), Ren, inter-renal (internal control) diameter (mm); Syst, peak systolic diameter (mm); Dias, end-diastolic diameter (mm); Veloc, radial wall velocity (mm/s); B, baseline; E, endpoint; TP, technical problem.

  • aOutlier in the analysis of radial wall velocity.

At the 4-week endpoint, seven of 15 non-irradiated control mice had aneurysmal dilatation in the suprarenal aneurysm-prone segments of the aorta that had progressed to ≥50% expansion (maximal CSD) over baseline, whereas none of the 13 low-level laser irradiated mice had this degree of additional progression of dilatation (P = 0.005 by Fisher's exact test) (Table 2 and Figures 2 and 3). Likewise, when repeating this analysis for ≥40% expansion and ≥30% expansion, we found that the incidence, in mice treated with LLLI, was also significantly lower than the non-irradiated control mice (P = 0.005 and P = 0.003, respectively, by Fisher's exact test).

Figure 2

Suprarenal AAA 4 weeks after angiotensin II infusion in the apolipoprotein E-deficient mouse (right) not present in similar mouse treated with low-level laser irradiation (left). The diameter of the needle standard is 0.5 mm.

Figure 3

High-frequency B-mode ultrasound measurements of the aortas of control and LLL-irradiated angiotensin II (Ang II)-infused apolipoprotein E-deficient mice at baseline and after 4 weeks. Note the prominent dilatation of the suprarenal aorta region 4 weeks after angiotensin infusion (left middle and centre middle) which is not present in the LLLI mouse (bottom panel). Arrows indicate medial dissection in the untreated suprarenal aortic aneurysm.

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Table 2

Aneurysmal dilatation in the suprarenal aneurysm-prone segments of the aorta over baseline

>50%>40%>30%
Control (n = 15)7a911
LLLI (n = 13)012
P0.0050.0050.003
  • aNumber of mice.

  • By Fisher's exact test.

At baseline (12–13 weeks of age), there was no significant difference in the mean maximum CSD of the suprarenal segments (normalized individually to the maximal CSD of the adjacent, non-aneurysm-prone inter-renal segment) between control and LLLI-treated animals (ratio of suprarenal/renal CSD, control vs. LLLI = 1.32 ± 0.11 vs. 1.29 ± 0.13, P = 0.5 by unpaired, two-tailed t-test) (Figure 4).

Figure 4

Effect of LLLI on aneurysmal dilatation of the suprarenal aneurysm-prone aortic segment of angiotensin II-infused apolipoprotein E-deficient mice. For this assessment, comparisons are performed between the mean (±SD) ratios of the maximal CSD of the suprarenal aortic segment normalized to those of the adjacent inter-renal segment for each group. Note the significant aortic expansion between baseline and endpoint in the control group (n = 15) (left) not seen in the LLLI-treated group (n = 13) (right). ***P < 0.001, by paired and unpaired, two-tailed t-test accordingly. White bars, baseline measurements; black bars, endpoint.

In the non-irradiated mice, the mean maximum CSD of the suprarenal aortic segments (normalized to the adjacent inter-renal aortic segments) increased significantly from baseline to 4 weeks (ratio of suprarenal/renal CSD, baseline vs. 4 weeks: 1.32 ± 0.11 vs. 1.82 ± 0.39, P = 0.0002 by paired, two-tailed t-test). However, in animals treated with LLLI, the maximum CSD of the suprarenal segments did not increase significantly from baseline to 4 weeks (mean of ratios of suprarenal/renal CSD, baseline vs. 4 weeks: 1.29 ± 0.13 vs. 1.32 ± 0.14, P = 0.49 by paired, two-tailed t-test).

Direct comparisons at the 4-week endpoint between the suprarenal segments of LLLI and non-irradiated animals (normalized individually to inter-renal control segments) showed a highly significant attenuating effect of LLLI on aneurysm progression in this model (mean of ratios of suprarenal/renal CSD, LLLI vs. non-irradiated: P = 0.0002 by unpaired, two-tailed t-test).

These results show an overall 94% reduction in maximum CSD of the suprarenal aneurysm-prone segments compared with baseline in the LLLI-treated mice in this model.

Analysis of the M-mode data (Figure 5 and Table 3) showed that the mean diastolic diameter and the mean systolic diameter were significantly higher in the control animals compared with LLLI-treated animals at the 4-week endpoint. In the non-treated control mice, the mean PD (normalized to systole) of the suprarenal aneurysm-prone aortic segments 4 weeks after angiotensin infusion was significantly lower than the mean PD at baseline. However, in the LLLI-treated mice, the mean PD at 4 weeks was not significantly different from baseline.

Figure 5

M-mode images of suprarenal aneurysm-prone segments showing marked decrease in RWV (slope) in the severely dilated aorta of the untreated mouse 4 weeks after angiotensin infusion (right) compared with baseline (upper left). This marked decrease in RWV is not seen in the aorta of the LLL-irradiated mouse (lower left). Note also the significant reduction in PD in the non-treated dilated aortic segment due to narrowing of the difference between peak systolic (S) and end-diastolic (D) diameter.

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Table 3

M-mode ultrasonographic measurements

ControlLLLILLLI vs. control at 4 weeks: P-value
Baseline4 weeksBaseline4 weeks
CSD in diastole (mm)1.10 ± 0.081.56 ± 0.351.03 ± 0.061.23 ± 0.21**0.007
CSD in systole (mm)1.24 ± 0.071.68 ± 0.311.18 ± 0.081.37 ± 0.18**0.005
PD0.11 ± 0.020.07 ± 0.05*0.13 ± 0.030.10 ± 0.04NS
RWV (mm/s)3.64 ± 0.892.34 ± 1.20**3.71 ± 0.733.98 ± 1.150.002
  • All parameters are expressed as means ± SD. Comparisons between measurements at baseline and at the 4-week endpoint were performed by paired, two-tailed t-test with the Bonferroni correction for multiple comparisons. Comparisons between control and LLLI mice at the 4-week endpoint were performed by unpaired, two-tailed t-test with the Bonferroni correction. Baseline vs. 4 weeks: *P < 0.05; **P < 0.01; P < 0.001. CSD, cross-sectional diameter; PD, pulse diameter (CSD in systole − CSD in diastole) normalized to CSD in systole; RWV, radial wall velocity.

When considering individual mice, at the 4-week endpoint, four of 14 non-irradiated control mice had >75% reduction in the PD (normalized to maximal diameter in systole) over baseline; whereas none of the 13 low-level laser irradiated mice had this degree of reduction in PD (P = 0.057 by Fisher's exact test).

The mean maximal RWV (Figure 6 and Table 3) of the control group was significantly lower at 4 weeks than at baseline, but no such difference in RWV was found between 4 weeks and baseline in the LLLI-treated mice. However, the mean RWV at 4 weeks was significantly greater in LLL-irradiated compared with the non-irradiated animals. Moreover, when considering individual mice, at the 4-week endpoint, six of 13 non-irradiated control mice had >50% reduction in the maximal RWV over baseline, whereas none of the 13 low-level laser irradiated mice had this degree of reduction of RWV (P = 0.007 by Fisher's exact test).

Figure 6

Effect of LLLI on RWV of the suprarenal aneurysm-prone aortic segment. Note that the mean (±SD) RWV in the non-treated control group (n = 13) was significantly lower at 4 weeks compared with baseline. However, there was a significant attenuation of this decrease in the LLL-irradiated mice (n = 13). **P < 0.01, by paired and unpaired, two-tailed t-test with the Bonferroni correction, respectively. White bars, baseline measurements; black bars, endpoint.

4. Discussion

We have shown that LLLI limits the progression of aneurysmal dilatation in the suprarenal aneurysm-prone segment of the abdominal aorta in angiotensin II-infused apolipoprotein E-deficient mice.

Whereas the higher power lasers used clinically for surgical excision and ablation convert photon energy into heat, low-level lasers cause only minor temperature elevations with the usual energy densities ranging between 0.1 and 4 J/cm2. A variety of studies have suggested that these low-energy photons are absorbed in the chromophores of the respiratory chain of the mitochondria.35 Studies by one of us (Lilach Gavish) have shown that this photon absorption apparently increases mitochondrial membrane potential.36 The latter is associated with an increase in the ATP energy store of the cell.37,38 This appears to represent a fundamental mechanism underlying the observed photomodulatory effects of LLLI.39

AAA is present in 6–10% of the population over the age of 65.40 Current forms of treatment are based on either open surgical repair with grafts or endovascular repair by large membrane-covered stents. Both techniques have major side effects with potentially life-threatening consequences2,3 emphasizing the importance of developing alternative therapeutic strategies, such as that presented in the current study, that target pathogenetic mechanisms of progression and rupture.

The angiotensin II-infused apolipoprotein E-deficient mouse model, developed by Daugherty et al.,27 shows important similarities to human AAA pathology. These include degradation of the elastic tissue associated with marked inflammatory cell infiltration and disruption of the musculo-elastic lamellar structure of the media including medial dissection.41 Similar changes have been detected in the elastase model of aneurysm42 and in the periarterial calcium chloride model developed in this laboratory.43,44 In the mouse angiotensin infusion model, these histological changes are usually found before the development of more advanced proliferative atherosclerotic lesions at these sites.27,41 However, AAA in humans, most commonly found in the infrarenal position, is usually diagnosed in a vessel that already has severe atherosclerotic changes in the wall. Nonetheless, marked inflammatory cell infiltration associated with disruption of the musculo-elastic architecture of the media is a major underlying histopathological process common to AAA in both animal and human models.

Measurements obtained by femoral artery catheter and the tail cuff method confirmed that angiotensin II infusion did not increase blood pressure in this model, and hence the suprarenal aneurysms, and the associated wall changes are considered to occur independently of this parameter.27

High-frequency ultrasonography (0.01 mm resolution) used in the current study was designed specifically for non-invasive microimaging in mice. Previous studies in the same mouse model showed a high correlation with direct post-mortem measurements of the outer diameter of the aorta obtained by digital photography30 and a very high correlation with morphometric measurements of H&E stained histological sections (r = 0.99).31 The measurements were also shown to have relatively small intra- and inter-individual variance.31 These studies support the accuracy and reproducibility of non-invasive high-resolution ultrasound monitoring of the dimensions of AAA in living mice including the effects of investigative manipulations and treatment regimens over time. Histology will be necessary to study effects of LLLI on cell and tissue morphology and pathobiology in this mouse model. However, the current study was designed to establish the effect of LLLI on aneurysm progression in vivo without the need for corrections of measurements for tissue shrinkage, embedding misalignment, or other perfusion fixation and preparation-related deformations of the arterial wall which confound accurate evaluation of wall shape by histopathology.

We have shown that in addition to a marked reduction in the mean maximum diameter of the suprarenal aortic segments of LLLI-treated vs. non-treated control aortas, the number of individual animals that developed aneurysmatic dilatation with >50% increase in maximum CSD over baseline was significantly lower in the laser-treated group. This degree of maximum diameter increase over baseline (or adjacent control segment) has been considered to be the accepted increment required for classification as a significant aneurysmal dilatation.27,31,41,43,44

Absolute measurements of CSD of the aorta can be considered sufficient for assessment of changes in aneurysmal dilatation from baseline to endpoint provided that both measurements are made in the same animal. However, when comparing the degree of aneurysmal dilatation in treated vs. non-treated animals, the use of absolute measurements alone fails to consider possible anatomical differences in vessel wall size between animals. Thus, in the current study, comparisons between treated and non-treated animals were performed after normalizing the diameter measurement of the suprarenal aneurysm-prone segment to that of the adjacent, non-aneurysm-prone, non-dilated, internal control, inter-renal segment whose aortic diameter has been shown not to change over 28 days in this model.31

Analysis of M-mode data showed that the mean PD of the suprarenal aneurysmatic segment of non-treated control mice decreased from baseline to the 4-week endpoint. However, this was less pronounced in the LLLI-treated animals, and the number of individual mice with significant reduction in PD was somewhat less (P = 0.057). Reduced PD is a reduction in the difference between the arterial wall diameter of the affected segment in systole and that in diastole. This occurs in situations of arterial wall weakening and/or reduced wall elasticity leading to reduced contractility such as that associated with aneurysmal dilatation.45 This is also consistent with data accumulated from a variety of studies including from our laboratory which have identified marked changes in matrix protein expression, secretion, and degradation in this and other models of aneurysm formation.

From M-mode data, we also found a significantly lower mean maximal RWV in control mice at the 4-week endpoint than that measured in the LLLI-treated mice, and a significantly greater number of control mice with >50% reduction in this parameter as compared with the LLL-treated mice. Maximal RWV is the first derivative, or slope, of the aortic diameter over time. Reduction in the maximal RWV occurs with a decrease in the speed of the wall motility during the cardiac cycle. This phenomenon presents as changes in the echogenic texture of the vessel wall. Such changes are seen in the cases of increased inflammatory cell infiltration that accompany changes in visco-elastic behaviour of the arterial wall.33,45 That mice treated with LLLI in the current study showed significantly less reduction in RWV than non-treated controls is consistent with our in vitro findings of the effects of this modality on expression and secretion of inflammatory chemokines and cytokines, the effects on cell proliferation and matrix protein secretion, and the known empiric effects on a variety of clinical entities where inflammation is a major pathogenetic substrate.

In conclusion, we have shown that LLLI significantly attenuates aneurysm formation in the angiotensin II-infused apolipoprotein E-deficient mouse. These studies, when considered together with previously reported in vitro studies,18,23 appear to provide strong support for initiation of studies in large animals as the next step towards testing the applicability of this technology to the human interventional setting.

Funding

These studies were supported in part by Swiss British Fiduciary Trust Center (Eliyahu Kelman, president), Rachel and Barney Gottstein Research Fund, Murray Koppelman Research Fund, and the Anna and Arnold Broniatovsky Research Fund of The Hebrew University of Jerusalem, Israel.

Acknowledgements

S.D.G. is the Brandman Foundation Professor of Cardiac and Pulmonary Diseases, The Hebrew University—Hadassah Medical School, Jerusalem, Israel. The authors gratefully acknowledge Professor Norman Grover for assistance with the statistical analysis.

Conflict of interest: none declared.

References

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