Christopher C. R. Bishop

Photodynamic Therapy of Normal and Balloon-Injured Rat Carotid Arteries Using 5-Amino-Levulinic Acid

BACKGROUND Although the management of atherosclerotic disease by the use of balloon angioplasty is widespread, the treatment is limited by restenosis in 30% to 50% of cases. Fibrocellular intimal hyperplasia, the main cause of restenosis, arises from proliferation and migration of medial smooth muscle cells (SMC) into the intimal layer. Factors leading to intimal hyperplasia are incompletely understood, and drugs have universally failed to influence clinical restenosis. Photodynamic therapy (PDT), the light activation of photosensitizing drugs to generate cytotoxic mediators, may have potential as prophylaxis for intimal hyperplasia. 5-Amino-levulinic acid-induced protoporphyrin IX (ALA-PPIX), a naturally occurring porphyrin precursor, and its product, -PPIX, offers a novel method of sensitization for PDT. We have investigated the pharmacokinetics of ALA in arteries and the effects of ALA-PPIX-sensitized PDT on normal and balloon-injured arteries. METHODS AND RESULTS ALA (20 to 200 mg/kg) was injected into healthy rats, and PPIX fluorescence was measured in the carotid arteries. In a second group of rats, the exposed carotid artery was laser illuminated (50 J/cm2, 630 nm) 30 to 90 minutes after sensitization. Three and 14 days after PDT, histological sections from treated arteries were analyzed by light microscopy. Subsequently, two new groups of rats underwent PDT (ALA, 100 mg/kg; laser, 50 J/cm2, 630 nm [at 60 to 90 minutes]). The left carotid arteries underwent balloon angioplasty by intraluminal passage of a Fogarty FG2 catheter immediately before irradiation. These rats were killed at 14 and 28 days together with laser-only, ALA-only, and untreated control rats. The arteries were perfusion-fixed in vivo. ALA-PPIX induced arterial media fluorescence in a dose-dependent manner. In the normal arteries, PDT produced a dose-dependent cellular depletion in the treated arterial segment at 3 days, and this was complete with 100 and 200 mg/kg of ALA. At 14 days, the media remained acellular, although the endothelial lining had regenerated. In the balloon-injured arteries, PDT produced complete inhibition of intimal hyperplasia at both 14 and 28 days (0%). This was significantly greater than that produced by any of the control rats (34% to 69% and 37% to 66% at the two times, respectively). Significance was at 99% using ANOVA and Fishers PLSD test. No hemorrhage, thrombosis, or aneurysm formation was seen. CONCLUSIONS ALA-PPIX-sensitized PDT applied at the time of angioplasty effectively inhibits experimental intimal hyperplasia development in rats. This may offer a new approach to the management of angioplasty restenosis in patients.

An Objective Characterization of Atherosclerotic Lesion An Alternative Method to Identify Unstable Plaque

BACKGROUND AND PURPOSE The aim of this study was to evaluate a computer-assisted technique to characterize atherosclerotic plaque. METHODS In 9 subjects (7 men, 2 women; mean age 33 years), known anatomic areas (carotid, fat, muscle, iliotibial tract, and tibia) were scanned with an Acuson duplex ultrasound machine with 72 machine settings. The ultrasound images of these anatomic areas were recorded on magneto-optical disks. Echo amplitude statistics were obtained, and the mean pixel value (MPV) was used to assess the level of echogenicity. The ideal settings of this particular scanner for optimal discrimination between these tissues types were identified by the Heuristic Index of Discrimination. With these settings, carotid artery scanning was performed on 17 patients (15 men, 2 women; mean age 65 years), and the image analysis of their ultrasound carotid plaques was compared with their histological findings. RESULTS In this study, discrimination between the selected tissues was found to be optimal when the controls were set at log 40 dB, 0/2/0, and gain of -5 dB. The MPV of the carotid specimens scanned at this setting correlated significantly with the histological findings (Spearman correlation, P=0.002). CONCLUSIONS Computer-assisted image analysis to give the MPV is a technique that may be used to identify unstable atherosclerotic plaques reliably.

Intra-arterial Photodynamic Therapy Using 5-ALA in a Swine Model

OBJECTIVES To test the hypothesis that intravascular light could be delivered via a balloon catheter for arterial photodynamic therapy (PDT). DESIGN Pig non-injury model. MATERIALS Clinical catheter equipment. METHODS Large White pigs (15-20 micrograms) were photosensitised with 5-aminolaevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) at a concentration of 120 mg/kg. Arterial biopsies were taken at intervals between 30 mins and 24 h and frozen sections analysed using a CCD camera to give a temporal profile of fluorescence in each arterial layer. PDT was given to normal arterial segments via a 4 mm transparent PTA balloon inflated so as to occlude flow, but not distend the artery. Animals were culled at 3 and 14 days and the above segments harvested. RESULTS Fluorescence peaked in the adventitia, intima and medial layers at 1.5, 4 and 6 h respectively. PDT at all time points produced VSMC depletion compared with controls. The degree of depletion mirrored the fluorescence profile of PpIX. CONCLUSIONS PDT can be delivered via a standard PTA balloon with a transparent channel. This depletes the VSMC population within the arterial wall without complications. Intra-arterial PDT is therefore a potential therapy to reduce the incidence of restenosis post-angioplasty.

Reduction in the response to coronary and iliac artery injury with photodynamic therapy using 5-aminolaevulinic acid

OBJECTIVE Photodynamic therapy (PDT) uses red light (non-thermal, non-ionising) to activate a previously administered photosensitizing drug. This inhibits neointimal hyperplasia in injured arteries in small animals where it appears safe and well tolerated. Our aim was to develop a method for percutaneous application of PDT to iliac and coronary arteries in a large animal model and investigate its influence on the remodeling and intimal hyperplastic response to balloon injury. METHODS Studies were undertaken on 13 juvenile Large White-Landrace crossbred pigs (15-20 kg). After intravenous administration of the photosensitizing agent 5-amino laevulinic acid (ALA), the arterial tree was accessed via the left common carotid artery and balloon injuries made by over-distension in both common iliacs (thirteen animals) and one or two main coronary arteries (eight animals). Half the injured sites were then illuminated with red laser light transmitted via the catheter. Animals were culled 28 days later and tissue harvested for histomorphometry. RESULTS Compared with control injured vessels, PDT treated, balloon injured coronary arteries had a larger lumen (1.4 vs. 0.8 mm2, P = 0.002), larger area within the external elastic lamina (2.8 vs. 2.2 mm2, P = 0.006) and smaller area of neointimal hyperplasia (0.4 vs. 0.7 mm2, P = 0.06), 28 days after intervention. Less neointimal hyperplasia and the absence of negative remodeling resulted in the lumen of PDT-treated, injured segments being the same as that of adjacent reference segments (1.5 vs. 1.6 mm2). Similar trends, but with smaller differences, were seen in the iliac vessels. CONCLUSIONS Intra-arterial, trans-catheter PDT favourably influences the arterial response to balloon injury in both the coronary and peripheral circulations. This technique offers a promising new approach to restenosis after endovascular procedures.

Inhibition of intimal hyperplasia in balloon injured arteries with adjunctive phthalocyanine sensitised photodynamic therapy

OBJECTIVES We investigated the effects of Photodynamic therapy (PDT) using Aluminium disulphonated phthalocyanine (AlS2Pc) on experimental intimal hyperplasia (FCIH). MATERIALS AND METHODS (a) Pharmacokinetics: Normal rats were injected with Als2Pc and carotid artery fluorescence was measured. (b) Normal artery PDT: Sensitised rats underwent carotid artery laser irradiation (50J/cm2, 675nm) and were assessed after 3 and 14 days and 1-6 months. (c) PDT: Rats underwent standard carotid artery balloon injury immediately prior to PDT and arteries were assessed at 2 to 26 weeks, together with laser, AlS2Pc, and untreated controls. CHIEF OUTCOME MEASURES (a) Fluorescence intensity in different arterial layers. (b) Medial smooth muscle cell counts per high power field (light microscopic). (c) Percentage amount of FCIH (area of intimal hyperplasia) as a ratio of the IEL (area enclosed by the internal elastic lamina). RESULTS (a) AlS2Pc fluorescence intensity increased with increasing dosage, with maximal fluorescence in the arterial media at 30 min. (b) PDT produced medial cell depletion at 3 days and persisted over 6 months without loss of vessel integrity. (c) PDT completely inhibited FCIH at 2 and 4 weeks. This was partial at 6 to 26 weeks (51% of untreated level). PDT inhibition of FCIH was significantly greater than in any of the control groups. p < 0.0001. Mann-Whitney Test. CONCLUSION Adjunctive AlS2Pc sensitised photodynamic therapy inhibits experimental intimal hyperplasia, by causing medial smooth muscle cell depletion. This offers a new approach to the management of angioplasty restenosis in patients.

NON-INVASIVE ASSESSMENT OF ARTERIAL STENOSES IN ANGIOPLASTY SURVEILLANCE :A COMPARISON WITH ANGIOGRAPHY

OBJECTIVES Comparison of non-invasive arterial measurements with angiography and their use for angioplasty surveillance. DESIGN Prospective assessments of arterial stenoses in patients undergoing angioplasty in a 9 month surveillance period. MATERIALS Fifty consecutive patients undergoing angioplasty. METHODS (i) One hundred and thirty-one sets of clinical assessments, ankle brachial Doppler pressure indices and colour Duplex velocities and diameters were compared to time-matched angiographic diameter stenosis. (ii) Fifty patients undergoing femoropopliteal angioplasty (32 stenoses and 18 occlusions) were studied with ankle branchial Doppler pressure indices and colour Duplex and angiography during a 9 month surveillance period. RESULTS (i) Symptoms, pulses, resting ABPI, and exercise ABPI showed no useful correlation with angiography. Duplex velocity ratio and Duplex diameters showed correlation and agreement with angiography respectively. (ii) On surveillance, restenosis was universal but not always clinically significant. Angioplasty caused a rapid improvement in ABPI and imaging studies which worsened at later times. ABPI did not predict clinical failure however, Duplex and angiography predicted all clinical failures. CONCLUSIONS Restenosis should be assessed with imaging of the angioplasty site during angioplasty surveillance.

Thrombosis and restenosis after peripheral angioplasty: Does acute 111indium-platelet accumulation predict angioplasty outcome?

OBJECTIVES Use of platelet deposition to predict failure following angioplasty. DESIGN Prospective study of angioplasty patients in a 9 months surveillance period. MATERIALS Thirty-eight successful angioplasty patients. METHODS Autologous 111indium-labelled platelets were re-injected immediately after angioplasty. Gamma camera and probe measures of radioactivity were obtained daily for 2-4 days and compared to a reference site to obtain a radioactivity ratio. Patient follow up was with duplex and arteriography from day 0 to 9 months or at angioplasty failure. RESULTS Thirty-one patients remained asymptomatic; two developed acute occlusion and five developed restenosis. Platelet accumulation (increased mean radioactivity ratio) occurred at all angioplasty sites and was significantly higher after acute occlusion (camera: 2.93 and probe: 1.93) compared to asymptomatic patients (camera: 1.25 and probe: 1.15) and restenotic patients (camera: 1.31 and probe: 1.23). Radioactivity ratio was not different in patients who later developed restenosis. CONCLUSION 111Indium platelet radioactivity effectively detected acute angioplasty reocclusions, but was unable to predict subsequent angioplasty restenosis.

Limitations of rat carotid balloon de-endothelialization model in arterial photodynamic therapy: a study using 5-aminolaevulinic acid

Photodynamic therapy (PDT) effectively inhibits fibrocellular intimal hyperplasia (FCIH) two and four weeks after arterial traction balloon injury in rat carotid arteries. The aim of the present study was to assess the long term effects of PDT in this rat model of FCIH. 5- aminolaevulinic acid-induced protoporphyrin IX was used to sensitize rats for PDT after traction balloon arterial injury to the whole of the left common carotid artery. Rats were sacrificed at intervals of six to 26 weeks, and perfusion fixed and H and E stained sections were analyzed using computerized morphometry. PDT inhibition of FCIH was only partial at these late times. The amount of FCIH present increased with increasing time after injury. The late occurrence of FCIH appeared to be due to migration of FCIH from balloon injured areas outside the PDT treated field as a result of the traction injury being applied to the whole carotid. We recommend segmental balloon injury rather than the traction injury of the whole common carotid injury for future studies in this model.

Pharmacokinetics and efficacy of 5-aminolaevulinic acid for endovascular photodynamic therapy in a swine model

Vascular smooth muscle cells (VSMC) are known to be important in restenosis and photodynamic therapy (PDT) has been shown experimentally to deplete the VSMC population in small animal studies. We aimed to investigate the pharmacokinetics of 5- aminolaevulinic acid (5-ALA) and to see if endovascular PDT was feasible in a large animal model. Large White pigs (15 - 20 Kg) were photosensitized with 5-ALA at a concentration of 60 and 120 mg/Kg. Arterial biopsies were taken at intervals between 30 mins and 24 hours and snap frozen in liquid nitrogen. Frozen sections were analyzed using a CCD camera and PpIX activity assessed by computing pixel counts for intima, media and adventitia at each time point. Based on the above results 8 pigs were treated with PDT at 1.5, 2.5 or 6 hours following 5-ALA administration. Iliac segments were then illuminated with 50 J/cm2, 635 nm wavelength light via a 4 mm transparent PTA balloon. Animals were culled at 3 days and the above segments pressure perfused in situ with 4% formyl saline, before being excised and fixed. Fluorescence peaked in the adventitia at 1.5 hours, was minimal at 2.5 hours and peaked in the media at 6 hours post 5-ALA. Of the second series of 8 pigs, all animals survived to culling and all treated arteries remained patent. Histological sections stained with H&E were examined and medial VSMCs counted in 4 individual high power fields per section. The mean VSMC count per HPF for PDT treated segments was 16, 51 and 12 at 1.5, 2.5 and 6 hours respectively. VSMC counts in ALA alone controls and light alone controls were 115 and 103 respectively (p less than 0.0001). Endovascular delivery of light to 5-ALA sensitized animals is therefore feasible and was not associated with any complications.

Photodynamic therapy of contractile arterial smooth muscle cells in vivo using aluminium disulphonated phthalocyanine: implications for angioplasty-induced intimal hyperplasia

Photodynamic therapy (PDT) of proliferative vascular smooth muscle cells (SMC) reduces intimal hyperplasia. We asses the effects of PDT on contractile SMC In normal arteries using aluminum disulphonated phthalocyanine (AlS2Pc) sensitization. Rats sensitized with intravenous AlS2Pc underwent carotid artery fluorescence measurement. More rats had carotid artery irradiation with different laser energies 30 minutes after sensitization with AlS2Pc 5.0 mg/kg and sacrificed at 3 days. Additional rats were irradiated with 50 J/cm2 after sensitization with different AlS2Pc doses and sacrificed at 3 and 14 days. Histological analysis was by light microscopy. Rats treated with PDT and sacrificed at 3 and 6 months were also analyzed. The results show AlS2Pc induced high and dose dependent fluorescence within the arterial media. All PDT light doses (50 - 250 J/cm2) produced arterial SMC depletion. In contrast, PDT sensitizer doses produced complete cell depletion only at high doses of AlS2Pc. These treated arteries were acellular after 3 days. At 14 days the media remained acellular but the endothelial lining regenerated. The arterial media remained acellular even at 6 months -- however, no hemorrhage, thrombosis or aneurysm formation was seen. Arterial PDT causes media contractile SMC depletion in vivo and the artery retains its structural and functional integrity.