Background The entomopathogenic fungus  Metarhizium anisopliae  is being considered as a biocontrol agent for adult African malaria vectors. In the laboratory, work was carried out to assess whether horizontal transmission of the pathogen can take place during copulation, as this would enhance the impact of the fungus on target populations when compared with insecticides.
Background
The entomopathogenic fungus  Metarhizium anisopliae  is being considered as a biocontrol agent for adult African malaria vectors. In the laboratory, work was carried out to assess whether horizontal transmission of the pathogen can take place during copulation, as this would enhance the impact of the fungus on target populations when compared with insecticides.Metarhizium anisopliaeMethods Virgin female  Anopheles gambiae sensu stricto  were exposed to conidia whilst resting on fungus-impregnated paper. These females were then placed together for one hour with uncontaminated males in proportions of either 1:1 or 1:10 shortly before the onset of mating activity.
Methods
Virgin female  Anopheles gambiae sensu stricto  were exposed to conidia whilst resting on fungus-impregnated paper. These females were then placed together for one hour with uncontaminated males in proportions of either 1:1 or 1:10 shortly before the onset of mating activity.Anopheles gambiae sensu strictoResults Males that had acquired fungal infection after mating indicate that passive transfer of the pathogen from infected females does occur, with mean male infection rates between 10.7 ± 3.2% and 33.3 ± 3.8%. The infections caused by horizontal transmission did not result in overall differences in survival between males from test and control groups, but in one of the three experiments the infected males had significantly shorter life spans than uninfected males ( P  < 0.05).
Results
Males that had acquired fungal infection after mating indicate that passive transfer of the pathogen from infected females does occur, with mean male infection rates between 10.7 ± 3.2% and 33.3 ± 3.8%. The infections caused by horizontal transmission did not result in overall differences in survival between males from test and control groups, but in one of the three experiments the infected males had significantly shorter life spans than uninfected males ( P  < 0.05).PConclusion This study shows that autodissemination of fungal inoculum between  An. gambiae s.s . mosquitoes during mating activity is possible under laboratory conditions. Field studies are required next, to assess the extent to which this phenomenon may augment the primary contamination pathway (i.e. direct contact with fungus-impregnated targets) of vector populations in the field.
Conclusion
This study shows that autodissemination of fungal inoculum between  An. gambiae s.s . mosquitoes during mating activity is possible under laboratory conditions. Field studies are required next, to assess the extent to which this phenomenon may augment the primary contamination pathway (i.e. direct contact with fungus-impregnated targets) of vector populations in the field.An. gambiae s.s

Introduction Control of the main African malaria vector  Anopheles gambiae  (Diptera: Culicidae) continues to rely heavily on application of residual insecticides, either for indoor residual house spraying [ 1 ] or bednet impregnation [ 2 ]. These approaches have been highly effective in reducing malaria morbidity and mortality [ 2 ], but associated problems regarding environmental pollution [ 3 , 4 ], acceptability and cost [ 5 , 6 ] and the now widespread and continuing development of resistance [ 7 - 10 ] underscore the need for alternative strategies, such as vector control with biological agents [ 1 , 11 , 12 ]. Entomopathogenic fungi are among the biological control agents used against insect pests. Interest in using the hyphomycete  Metarhizium anisopliae  against adult African malaria vectors has recently increased [ 13 ]. The fungus has proven to be highly virulent for this vector, both in the laboratory [ 14 ] as well as in the field (Scholte  et al ., in preparation). The principal method of contamination of the target insect population with the fungus is through application of conidia on indoor resting targets. However, in order to achieve the highest possible impact on the target population, it is desirable that contamination pathways other than the primary mode of contamination are utilised, for instance horizontal transmission. Horizontal transmission of pathogens within the same host/target species is called autodissemination, and this phenomenon has been suggested for biocontrol of several insect pests [ 15 , 16 ]. Successful transmission of  M. anisopliae  by honeybees for infection of the pollen beetle  Meligethes aeneus  [ 17 ], of  Beauveria bassiana  between adult flies of  Delia radicum  [ 18 ] and of  M. anisopliae  and  B. bassiana  between adult tsetse flies,  Glossina morsitans morsitans  [ 19 ] confirms the capability of insects to transmit fungi horizontally. Autodissemination of insecticidal biocontrol agents, such as insect-pathogenic fungi, provides an additional advantage over pesticides, as the impact on pest populations increases beyond direct contact. In several cases, autodissemination of entomopathogenic fungi within populations of insect pests, using attractant traps as the initial source of infection, has succeeded [ 18 , 20 - 22 ]. The strategy envisaged for the use of  M. anisopliae  against adult  An. gambiae  is that host-seeking females, and occasionally also males that rest indoors, will receive primary infections while resting indoors on fungus-impregnated resting targets. Under optimal circumstances, prior to death, this infection may be transmitted to conspecifics upon contact (e.g. during mating). These mosquitoes are, therefore, not infected through direct contact with fungus-impregnated materials, but indirectly, upon physical contact with infected counterparts. It is estimated that approximately half of newly hatched, virgin females take a blood meal before mating [ 23 , 24 ]. A female, with contaminated legs and mouthparts following the blood-feeding visit to a house containing fungus-impregnated resting targets, may contaminate male counterparts when she mates the following dusk period, thereby spreading the fungus through the population. The objective of this study was to investigate whether adult  An. gambiae  infected with  M. anisopliae  can transmit the fungus to uncontaminated mosquitoes of the opposite sex through physical contact during the mating process.
Introduction
Control of the main African malaria vector  Anopheles gambiae  (Diptera: Culicidae) continues to rely heavily on application of residual insecticides, either for indoor residual house spraying [ 1 ] or bednet impregnation [ 2 ]. These approaches have been highly effective in reducing malaria morbidity and mortality [ 2 ], but associated problems regarding environmental pollution [ 3 , 4 ], acceptability and cost [ 5 , 6 ] and the now widespread and continuing development of resistance [ 7 - 10 ] underscore the need for alternative strategies, such as vector control with biological agents [ 1 , 11 , 12 ].Anopheles gambiaeEntomopathogenic fungi are among the biological control agents used against insect pests. Interest in using the hyphomycete  Metarhizium anisopliae  against adult African malaria vectors has recently increased [ 13 ]. The fungus has proven to be highly virulent for this vector, both in the laboratory [ 14 ] as well as in the field (Scholte  et al ., in preparation). The principal method of contamination of the target insect population with the fungus is through application of conidia on indoor resting targets. However, in order to achieve the highest possible impact on the target population, it is desirable that contamination pathways other than the primary mode of contamination are utilised, for instance horizontal transmission. Horizontal transmission of pathogens within the same host/target species is called autodissemination, and this phenomenon has been suggested for biocontrol of several insect pests [ 15 , 16 ]. Successful transmission of  M. anisopliae  by honeybees for infection of the pollen beetle  Meligethes aeneus  [ 17 ], of  Beauveria bassiana  between adult flies of  Delia radicum  [ 18 ] and of  M. anisopliae  and  B. bassiana  between adult tsetse flies,  Glossina morsitans morsitans  [ 19 ] confirms the capability of insects to transmit fungi horizontally. Autodissemination of insecticidal biocontrol agents, such as insect-pathogenic fungi, provides an additional advantage over pesticides, as the impact on pest populations increases beyond direct contact. In several cases, autodissemination of entomopathogenic fungi within populations of insect pests, using attractant traps as the initial source of infection, has succeeded [ 18 , 20 - 22 ]. The strategy envisaged for the use of  M. anisopliae  against adult  An. gambiae  is that host-seeking females, and occasionally also males that rest indoors, will receive primary infections while resting indoors on fungus-impregnated resting targets. Under optimal circumstances, prior to death, this infection may be transmitted to conspecifics upon contact (e.g. during mating). These mosquitoes are, therefore, not infected through direct contact with fungus-impregnated materials, but indirectly, upon physical contact with infected counterparts. It is estimated that approximately half of newly hatched, virgin females take a blood meal before mating [ 23 , 24 ]. A female, with contaminated legs and mouthparts following the blood-feeding visit to a house containing fungus-impregnated resting targets, may contaminate male counterparts when she mates the following dusk period, thereby spreading the fungus through the population.Metarhizium anisopliaeet alM. anisopliaeMeligethes aeneusBeauveria bassianaDelia radicumM. anisopliaeB. bassianaGlossina morsitans morsitansM. anisopliaeAn. gambiaeThe objective of this study was to investigate whether adult  An. gambiae  infected with  M. anisopliae  can transmit the fungus to uncontaminated mosquitoes of the opposite sex through physical contact during the mating process.An. gambiaeM. anisopliaeMaterials and Methods Bioassays Three bioassays were conducted to assess whether autodissemination of  M. anisopliae  can occur during the process of mating: 1) 30 fungus-contaminated virgin females and 30 uncontaminated males were placed in a standard (30 cm 3 ) netting cage for one hour during the normal mating period; 2) a single fungus-contaminated virgin female and a single uncontaminated male where placed together in a glass tube for one hour during the normal mating period; 3) a single fungus-contaminated virgin female and 10 uncontaminated males were placed in a standard (30 cm 3 ) netting cage for one hour during the normal mating period. A fourth bioassay assessed whether or not: 4) males that became infected with the fungus had acquired the infective propagules from contaminated females or rather from contact with the substrate where those females had rested previously. Mosquitoes The  Anopheles gambiae sensu stricto  strain used originates from Suakoko, Liberia (courtesy Prof. M. Coluzzi) and has been maintained in the Wageningen laboratories since 1989, under standardized conditions described by Mukabana  et al . [ 25 ]. Experimental mosquitoes were four (females) or seven day old (males). Virginity of the females was assured by collecting them within 24 hrs after emergence from cages and keeping them separate from the males prior to the experiments. In all experiments, mosquito mortality was checked daily, the mosquito cadavers placed on moist filter paper and placed in Petri dishes that were sealed with parafilm. These were kept in an incubator at 27 ± 2°C and checked for fungal sporulation after three days. Fungus Metarhizium anisopliae  var.  anisopliae  isolate ICIPE30 (courtesy Dr. N.K. Maniania) was originally isolated in 1989 from a stemborer,  Busseola fusca  Fuller, near Kendu Bay, Western Kenya. Conidia were inoculated on oatmeal agar (OA) and placed in an incubator to grow. Fungal virulence was maintained by passing it through  An. gambiae  mosquitoes every five cycles after growing on OA. Third instar larvae were infected by applying dry conidia on the water surface. After 48–72 hours, moribund larvae were removed and their thorax opened to remove tissue with blastospores. These were plated on OA and placed in a dark incubator at 27°C. One week after the onset of sporulation of the colonies, conidia were harvested using a 0.05% Triton-X solution and a glass rod. The solvent with conidia was concentrated by removing the supernatant after centrifuging for three minutes at 5000 rpm. Dilutions were made using 0.05% Tween80 to obtain a conidial concentration of 10 8  conidia ml -1 . Vegetable (sunflower) oil was added to obtain an 8% adjuvant oil formulation. Five ml of this suspension was pipetted evenly over a 240 cm 2  piece of filter paper resulting in conidial densities of 1.6 × 10 10  conidia m -2 . The impregnated paper was left to dry at 20°C and 75 ± 5% RH for 48 hours and was then placed on the inside of a plastic cylinder (height 11.3 cm, diameter 3.4 cm) in such manner that the paper neatly lined the upright wall of the tube. The top of the tube was covered with netting material. This setup was used only to infect female mosquitoes. Before any contamination, the viability of the impregnated conidia was checked by placing a 1 cm 2  piece of the impregnated paper on a Sabourad Dextrose Agar in an incubator at 27°C in the dark for 16–20 hours. After incubation, the piece of paper was carefully removed and placed under a microscope (X 400) to determine the proportion germinated. For direct contamination of the female mosquitoes with  M. anisopliae , around 30 individuals were placed in the cylinder for 24 hours. Experimental procedures Bioassay 1 Thirty uninfected males were placed in a 30 cm 3  netting cage three hours before the onset of mating. Half an hour before (artificial) dusk, which for  An. gambiae  is the time when mating activity occurs [ 26 ], thirty contaminated females were added to this cage by releasing them from the cylinder (see above) where they had spent the previous 24 hours. By that time a large percentage of the males had the fibrillae on their antennae erect, which is considered a sign for impending mating activity [ 27 ]. One hour after introduction of the females, all males were gently removed from the cage using a 2 cm diameter glass vial and placed in a clean cage where they had access to 6% glucose  ad libitum . The experiment was replicated three times, on different days. Control groups were treated similarly, with the difference that the paper lining the contamination tube was void of conidia. Mortality of males and females was checked daily to measure longevity. Dead mosquitoes (both sexes) were removed from the cages and placed on moist filter paper in a Petri dish, which was sealed and examined for fungal growth three days later. An additional similar experiment with only five females and five males was performed to determine whether conidia could be seen on the cuticle of the test males directly after the females were removed. Males were killed by a droplet of chloroform and placed under a microscope (X 400) and examined for attached conidia. Bioassay 2 A single, uncontaminated 7-day old male was placed in a clean glass vial (diameter 2 cm, height 10 cm, sealed off with netting), to which one  M. anisopliae  contaminated female was added 30 minutes before the onset of mating activity. After one hour the couple was separated by gently removing the male, which was placed and kept in a separate glass vial until it died. A wad of cotton wool moist with 6% glucose was placed on top of the vial. Females remained in the vial and were provided with glucose in the same way. Mortality of both sexes was recorded daily. The control group consisted of an equal number of pairs that were handled equally, with the difference that the females were not infected with the fungus. This was repeated with 35 male-female pairs on three different days. Bioassay 3 Ten uncontaminated 7-day old males were placed in a 30 cm 3  netting cage. Half an hour before the onset of mating activity, a single infected female mosquito was added to this cage with the males. After one hour each male was gently removed using a clean 2 cm diameter glass vial and kept alive individually as in bioassay 2. This was done 14 times for the test group and six times for the control group. Bioassay 4 To assess whether the males in the above bioassays had acquired fungal infection from the contaminated females during the process of mating, or from resting on the substrate where fungus-contaminated females had rested previously (glass and netting), two experiments were carried out. In one experiment a total of 46 contaminated females (in two separate trials) were gently transferred to a 500 ml glass beaker, sealed of with a glass Petri-dish. The females were killed after one hour by applying a droplet of chloroform in the beaker. They were then removed and placed into a Petri-dish on a piece of moist filter paper. The dish was sealed with parafilm, to be checked three days later for fungal infection. Directly after removal of the contaminated females, a total of 47 uncontaminated males (in two separate trials) were placed in the beaker for three days, after which they were killed, removed and checked for fungal infection as described above. During the three days inside the beaker they had continuous access to a cotton wool wad moist with a 6% glucose solution. The second experiment differed only in that a standard netting cage was used instead of a glass beaker, with 64 fungus-contaminated females and 67 uncontaminated males in two separate trials. Data analysis Mortality data were subjected to Kaplan-Meier pair-wise comparison survival analysis. Mosquito mortality data closely fitted the Gompertz distribution [ 28 ]. Comparisons of the average ages at the time of death were calculated and analyzed using GLM analysis. All analyses were carried out by using Genstat 7.0.
Materials and Methods
Bioassays Three bioassays were conducted to assess whether autodissemination of  M. anisopliae  can occur during the process of mating: 1) 30 fungus-contaminated virgin females and 30 uncontaminated males were placed in a standard (30 cm 3 ) netting cage for one hour during the normal mating period; 2) a single fungus-contaminated virgin female and a single uncontaminated male where placed together in a glass tube for one hour during the normal mating period; 3) a single fungus-contaminated virgin female and 10 uncontaminated males were placed in a standard (30 cm 3 ) netting cage for one hour during the normal mating period. A fourth bioassay assessed whether or not: 4) males that became infected with the fungus had acquired the infective propagules from contaminated females or rather from contact with the substrate where those females had rested previously.
Bioassays
Three bioassays were conducted to assess whether autodissemination of  M. anisopliae  can occur during the process of mating:M. anisopliae1) 30 fungus-contaminated virgin females and 30 uncontaminated males were placed in a standard (30 cm 3 ) netting cage for one hour during the normal mating period;2) a single fungus-contaminated virgin female and a single uncontaminated male where placed together in a glass tube for one hour during the normal mating period;3) a single fungus-contaminated virgin female and 10 uncontaminated males were placed in a standard (30 cm 3 ) netting cage for one hour during the normal mating period.A fourth bioassay assessed whether or not:4) males that became infected with the fungus had acquired the infective propagules from contaminated females or rather from contact with the substrate where those females had rested previously.Mosquitoes The  Anopheles gambiae sensu stricto  strain used originates from Suakoko, Liberia (courtesy Prof. M. Coluzzi) and has been maintained in the Wageningen laboratories since 1989, under standardized conditions described by Mukabana  et al . [ 25 ]. Experimental mosquitoes were four (females) or seven day old (males). Virginity of the females was assured by collecting them within 24 hrs after emergence from cages and keeping them separate from the males prior to the experiments. In all experiments, mosquito mortality was checked daily, the mosquito cadavers placed on moist filter paper and placed in Petri dishes that were sealed with parafilm. These were kept in an incubator at 27 ± 2°C and checked for fungal sporulation after three days.
Mosquitoes
The  Anopheles gambiae sensu stricto  strain used originates from Suakoko, Liberia (courtesy Prof. M. Coluzzi) and has been maintained in the Wageningen laboratories since 1989, under standardized conditions described by Mukabana  et al . [ 25 ]. Experimental mosquitoes were four (females) or seven day old (males). Virginity of the females was assured by collecting them within 24 hrs after emergence from cages and keeping them separate from the males prior to the experiments. In all experiments, mosquito mortality was checked daily, the mosquito cadavers placed on moist filter paper and placed in Petri dishes that were sealed with parafilm. These were kept in an incubator at 27 ± 2°C and checked for fungal sporulation after three days.Anopheles gambiae sensu strictoet alFungus Metarhizium anisopliae  var.  anisopliae  isolate ICIPE30 (courtesy Dr. N.K. Maniania) was originally isolated in 1989 from a stemborer,  Busseola fusca  Fuller, near Kendu Bay, Western Kenya. Conidia were inoculated on oatmeal agar (OA) and placed in an incubator to grow. Fungal virulence was maintained by passing it through  An. gambiae  mosquitoes every five cycles after growing on OA. Third instar larvae were infected by applying dry conidia on the water surface. After 48–72 hours, moribund larvae were removed and their thorax opened to remove tissue with blastospores. These were plated on OA and placed in a dark incubator at 27°C. One week after the onset of sporulation of the colonies, conidia were harvested using a 0.05% Triton-X solution and a glass rod. The solvent with conidia was concentrated by removing the supernatant after centrifuging for three minutes at 5000 rpm. Dilutions were made using 0.05% Tween80 to obtain a conidial concentration of 10 8  conidia ml -1 . Vegetable (sunflower) oil was added to obtain an 8% adjuvant oil formulation. Five ml of this suspension was pipetted evenly over a 240 cm 2  piece of filter paper resulting in conidial densities of 1.6 × 10 10  conidia m -2 . The impregnated paper was left to dry at 20°C and 75 ± 5% RH for 48 hours and was then placed on the inside of a plastic cylinder (height 11.3 cm, diameter 3.4 cm) in such manner that the paper neatly lined the upright wall of the tube. The top of the tube was covered with netting material. This setup was used only to infect female mosquitoes. Before any contamination, the viability of the impregnated conidia was checked by placing a 1 cm 2  piece of the impregnated paper on a Sabourad Dextrose Agar in an incubator at 27°C in the dark for 16–20 hours. After incubation, the piece of paper was carefully removed and placed under a microscope (X 400) to determine the proportion germinated. For direct contamination of the female mosquitoes with  M. anisopliae , around 30 individuals were placed in the cylinder for 24 hours.
Fungus
Metarhizium anisopliae  var.  anisopliae  isolate ICIPE30 (courtesy Dr. N.K. Maniania) was originally isolated in 1989 from a stemborer,  Busseola fusca  Fuller, near Kendu Bay, Western Kenya. Conidia were inoculated on oatmeal agar (OA) and placed in an incubator to grow. Fungal virulence was maintained by passing it through  An. gambiae  mosquitoes every five cycles after growing on OA. Third instar larvae were infected by applying dry conidia on the water surface. After 48–72 hours, moribund larvae were removed and their thorax opened to remove tissue with blastospores. These were plated on OA and placed in a dark incubator at 27°C. One week after the onset of sporulation of the colonies, conidia were harvested using a 0.05% Triton-X solution and a glass rod. The solvent with conidia was concentrated by removing the supernatant after centrifuging for three minutes at 5000 rpm. Dilutions were made using 0.05% Tween80 to obtain a conidial concentration of 10 8  conidia ml -1 . Vegetable (sunflower) oil was added to obtain an 8% adjuvant oil formulation. Five ml of this suspension was pipetted evenly over a 240 cm 2  piece of filter paper resulting in conidial densities of 1.6 × 10 10  conidia m -2 . The impregnated paper was left to dry at 20°C and 75 ± 5% RH for 48 hours and was then placed on the inside of a plastic cylinder (height 11.3 cm, diameter 3.4 cm) in such manner that the paper neatly lined the upright wall of the tube. The top of the tube was covered with netting material. This setup was used only to infect female mosquitoes. Before any contamination, the viability of the impregnated conidia was checked by placing a 1 cm 2  piece of the impregnated paper on a Sabourad Dextrose Agar in an incubator at 27°C in the dark for 16–20 hours. After incubation, the piece of paper was carefully removed and placed under a microscope (X 400) to determine the proportion germinated. For direct contamination of the female mosquitoes with  M. anisopliae , around 30 individuals were placed in the cylinder for 24 hours.Metarhizium anisopliaeanisopliaeBusseola fuscaAn. gambiaeM. anisopliaeExperimental procedures Bioassay 1 Thirty uninfected males were placed in a 30 cm 3  netting cage three hours before the onset of mating. Half an hour before (artificial) dusk, which for  An. gambiae  is the time when mating activity occurs [ 26 ], thirty contaminated females were added to this cage by releasing them from the cylinder (see above) where they had spent the previous 24 hours. By that time a large percentage of the males had the fibrillae on their antennae erect, which is considered a sign for impending mating activity [ 27 ]. One hour after introduction of the females, all males were gently removed from the cage using a 2 cm diameter glass vial and placed in a clean cage where they had access to 6% glucose  ad libitum . The experiment was replicated three times, on different days. Control groups were treated similarly, with the difference that the paper lining the contamination tube was void of conidia. Mortality of males and females was checked daily to measure longevity. Dead mosquitoes (both sexes) were removed from the cages and placed on moist filter paper in a Petri dish, which was sealed and examined for fungal growth three days later. An additional similar experiment with only five females and five males was performed to determine whether conidia could be seen on the cuticle of the test males directly after the females were removed. Males were killed by a droplet of chloroform and placed under a microscope (X 400) and examined for attached conidia. Bioassay 2 A single, uncontaminated 7-day old male was placed in a clean glass vial (diameter 2 cm, height 10 cm, sealed off with netting), to which one  M. anisopliae  contaminated female was added 30 minutes before the onset of mating activity. After one hour the couple was separated by gently removing the male, which was placed and kept in a separate glass vial until it died. A wad of cotton wool moist with 6% glucose was placed on top of the vial. Females remained in the vial and were provided with glucose in the same way. Mortality of both sexes was recorded daily. The control group consisted of an equal number of pairs that were handled equally, with the difference that the females were not infected with the fungus. This was repeated with 35 male-female pairs on three different days. Bioassay 3 Ten uncontaminated 7-day old males were placed in a 30 cm 3  netting cage. Half an hour before the onset of mating activity, a single infected female mosquito was added to this cage with the males. After one hour each male was gently removed using a clean 2 cm diameter glass vial and kept alive individually as in bioassay 2. This was done 14 times for the test group and six times for the control group. Bioassay 4 To assess whether the males in the above bioassays had acquired fungal infection from the contaminated females during the process of mating, or from resting on the substrate where fungus-contaminated females had rested previously (glass and netting), two experiments were carried out. In one experiment a total of 46 contaminated females (in two separate trials) were gently transferred to a 500 ml glass beaker, sealed of with a glass Petri-dish. The females were killed after one hour by applying a droplet of chloroform in the beaker. They were then removed and placed into a Petri-dish on a piece of moist filter paper. The dish was sealed with parafilm, to be checked three days later for fungal infection. Directly after removal of the contaminated females, a total of 47 uncontaminated males (in two separate trials) were placed in the beaker for three days, after which they were killed, removed and checked for fungal infection as described above. During the three days inside the beaker they had continuous access to a cotton wool wad moist with a 6% glucose solution. The second experiment differed only in that a standard netting cage was used instead of a glass beaker, with 64 fungus-contaminated females and 67 uncontaminated males in two separate trials.
Experimental procedures
Bioassay 1 Thirty uninfected males were placed in a 30 cm 3  netting cage three hours before the onset of mating. Half an hour before (artificial) dusk, which for  An. gambiae  is the time when mating activity occurs [ 26 ], thirty contaminated females were added to this cage by releasing them from the cylinder (see above) where they had spent the previous 24 hours. By that time a large percentage of the males had the fibrillae on their antennae erect, which is considered a sign for impending mating activity [ 27 ]. One hour after introduction of the females, all males were gently removed from the cage using a 2 cm diameter glass vial and placed in a clean cage where they had access to 6% glucose  ad libitum . The experiment was replicated three times, on different days. Control groups were treated similarly, with the difference that the paper lining the contamination tube was void of conidia. Mortality of males and females was checked daily to measure longevity. Dead mosquitoes (both sexes) were removed from the cages and placed on moist filter paper in a Petri dish, which was sealed and examined for fungal growth three days later. An additional similar experiment with only five females and five males was performed to determine whether conidia could be seen on the cuticle of the test males directly after the females were removed. Males were killed by a droplet of chloroform and placed under a microscope (X 400) and examined for attached conidia.
Bioassay 1
Thirty uninfected males were placed in a 30 cm 3  netting cage three hours before the onset of mating. Half an hour before (artificial) dusk, which for  An. gambiae  is the time when mating activity occurs [ 26 ], thirty contaminated females were added to this cage by releasing them from the cylinder (see above) where they had spent the previous 24 hours. By that time a large percentage of the males had the fibrillae on their antennae erect, which is considered a sign for impending mating activity [ 27 ]. One hour after introduction of the females, all males were gently removed from the cage using a 2 cm diameter glass vial and placed in a clean cage where they had access to 6% glucose  ad libitum . The experiment was replicated three times, on different days. Control groups were treated similarly, with the difference that the paper lining the contamination tube was void of conidia. Mortality of males and females was checked daily to measure longevity. Dead mosquitoes (both sexes) were removed from the cages and placed on moist filter paper in a Petri dish, which was sealed and examined for fungal growth three days later. An additional similar experiment with only five females and five males was performed to determine whether conidia could be seen on the cuticle of the test males directly after the females were removed. Males were killed by a droplet of chloroform and placed under a microscope (X 400) and examined for attached conidia.An. gambiaead libitumBioassay 2 A single, uncontaminated 7-day old male was placed in a clean glass vial (diameter 2 cm, height 10 cm, sealed off with netting), to which one  M. anisopliae  contaminated female was added 30 minutes before the onset of mating activity. After one hour the couple was separated by gently removing the male, which was placed and kept in a separate glass vial until it died. A wad of cotton wool moist with 6% glucose was placed on top of the vial. Females remained in the vial and were provided with glucose in the same way. Mortality of both sexes was recorded daily. The control group consisted of an equal number of pairs that were handled equally, with the difference that the females were not infected with the fungus. This was repeated with 35 male-female pairs on three different days.
Bioassay 2
A single, uncontaminated 7-day old male was placed in a clean glass vial (diameter 2 cm, height 10 cm, sealed off with netting), to which one  M. anisopliae  contaminated female was added 30 minutes before the onset of mating activity. After one hour the couple was separated by gently removing the male, which was placed and kept in a separate glass vial until it died. A wad of cotton wool moist with 6% glucose was placed on top of the vial. Females remained in the vial and were provided with glucose in the same way. Mortality of both sexes was recorded daily. The control group consisted of an equal number of pairs that were handled equally, with the difference that the females were not infected with the fungus. This was repeated with 35 male-female pairs on three different days.M. anisopliaeBioassay 3 Ten uncontaminated 7-day old males were placed in a 30 cm 3  netting cage. Half an hour before the onset of mating activity, a single infected female mosquito was added to this cage with the males. After one hour each male was gently removed using a clean 2 cm diameter glass vial and kept alive individually as in bioassay 2. This was done 14 times for the test group and six times for the control group.
Bioassay 3
Ten uncontaminated 7-day old males were placed in a 30 cm 3  netting cage. Half an hour before the onset of mating activity, a single infected female mosquito was added to this cage with the males. After one hour each male was gently removed using a clean 2 cm diameter glass vial and kept alive individually as in bioassay 2. This was done 14 times for the test group and six times for the control group.Bioassay 4 To assess whether the males in the above bioassays had acquired fungal infection from the contaminated females during the process of mating, or from resting on the substrate where fungus-contaminated females had rested previously (glass and netting), two experiments were carried out. In one experiment a total of 46 contaminated females (in two separate trials) were gently transferred to a 500 ml glass beaker, sealed of with a glass Petri-dish. The females were killed after one hour by applying a droplet of chloroform in the beaker. They were then removed and placed into a Petri-dish on a piece of moist filter paper. The dish was sealed with parafilm, to be checked three days later for fungal infection. Directly after removal of the contaminated females, a total of 47 uncontaminated males (in two separate trials) were placed in the beaker for three days, after which they were killed, removed and checked for fungal infection as described above. During the three days inside the beaker they had continuous access to a cotton wool wad moist with a 6% glucose solution. The second experiment differed only in that a standard netting cage was used instead of a glass beaker, with 64 fungus-contaminated females and 67 uncontaminated males in two separate trials.
Bioassay 4
To assess whether the males in the above bioassays had acquired fungal infection from the contaminated females during the process of mating, or from resting on the substrate where fungus-contaminated females had rested previously (glass and netting), two experiments were carried out. In one experiment a total of 46 contaminated females (in two separate trials) were gently transferred to a 500 ml glass beaker, sealed of with a glass Petri-dish. The females were killed after one hour by applying a droplet of chloroform in the beaker. They were then removed and placed into a Petri-dish on a piece of moist filter paper. The dish was sealed with parafilm, to be checked three days later for fungal infection. Directly after removal of the contaminated females, a total of 47 uncontaminated males (in two separate trials) were placed in the beaker for three days, after which they were killed, removed and checked for fungal infection as described above. During the three days inside the beaker they had continuous access to a cotton wool wad moist with a 6% glucose solution. The second experiment differed only in that a standard netting cage was used instead of a glass beaker, with 64 fungus-contaminated females and 67 uncontaminated males in two separate trials.Data analysis Mortality data were subjected to Kaplan-Meier pair-wise comparison survival analysis. Mosquito mortality data closely fitted the Gompertz distribution [ 28 ]. Comparisons of the average ages at the time of death were calculated and analyzed using GLM analysis. All analyses were carried out by using Genstat 7.0.
Data analysis
Mortality data were subjected to Kaplan-Meier pair-wise comparison survival analysis. Mosquito mortality data closely fitted the Gompertz distribution [ 28 ]. Comparisons of the average ages at the time of death were calculated and analyzed using GLM analysis. All analyses were carried out by using Genstat 7.0.Results In all four bioassays, female mosquitoes that had been exposed to conidia in the cylinder setup died significantly faster than the control females (F = 104.4; p < 0.001), with an average of 96.4 ± 2.0% of the cadavers having sporulating  M. anisopliae . All three replicates of bioassay 1 indicated transfer of the fungus from contaminated females to uninfected males, with an average male infection rate of 26.1 ± 5.3% (Table  1 ). There was no difference in survival between the males of the control and test groups (F = 0.30; p = 0.5844), but when the group of test males was split into those that had been infected and those that had not, survival analysis showed a difference in survival approaching significance (F = 2.73; p = 0.098). Under a compound microscope, conidia were observed on four of the five males. Most were found on the lower parts (tibia, tarsi, uncinus (claws) and arolium) of the first and second pair of legs (Figure  1 ). A few conidia were found attached to the hairs of the tip of the wings. No conidia were found on the head, thorax, abdomen or the hind legs. Per mosquito 0–25 conidia were found. Female mosquitoes that had spent 24 hrs on fungus-impregnated paper had conidia on legs, tips of their wings and mouthparts, but not on the thorax or abdomen. Table 1 Autodissemation of  M. anisopliae  from females to males  An. gambiae s.s .. The proportions of male mosquitoes infected are shown with the differences in survival of fungus-infected males compared to uninfected males within the test groups. Bioassay Ratio Average %(± SEM a ) of males infected Survival analysis (Kaplan Meier) Average age of males at time of death (days) infected uninfected 1 1:1 26.1 ± 5.3 F = 2.73; p = 0.098 10.3 ± 0.7 18.6 ± 0.8 b 2 1:1 34.0 ± 0.6 F = 3.39; p = 0.065 11.3 ± 1.9 15.6 ± 1.6 b 3 1:10 10.7 ± 3.2 F = 13.02; p = 0.001 13.1 ± 1.3 17.9 ± 0.6 b a : SEM = standard error of mean b : Significant difference (p < 0.05) between infected and uninfected males using GLM analysis (Genstat 7.0). Figure 1 Conidia of the entomopathogenic fungus  Metarhizium anisopliae  on the lower part of a male  Anopheles gambiae s.s . leg, after having been horizontally transferred from an infected female during copulation. In the second bioassay, with individual pairs in glass vials, an average of 34.2 ± 0.6% of the males acquired and died of the fungal infection. Survival of these males as a group was not significantly different from the control group (F = 1.27; p = 0.259). When the males from the test group were split into those that became infected and those that did not, a decrease in survival of the infected test males compared to the uninfected males was observed, although this effect was not significant (F = 3.39; p = 0.065). However, the average age at death of 11.3 ± 1.9 days of the fungus-infected males was significantly lower than the 15.6 ± 1.6 days of the uninfected test males (p = 0.001). In the third bioassay, horizontal transmission occurred in nine of the 14 replicates (64.3%). In those nine trials an average of 16.7 ± 3.7% of the 10 males acquired an infection. Calculated over the 14 trials, the infection rate was 10.7 ± 3.2%. As in the other two bioassays, there was no significant difference in survival rates between the test and control groups (F = 2.19; p = 0.139). However, those males in the test group that were found with sporulating fungus died significantly faster than uninfected males (F = 13.02; p = 0.001) with the average age at the time of death of 8.4 ± 1.1 days for infected and 17.9 ± 0.6 days for uninfected males, respectively. None of the 114 males in bioassay 4 were found infected with the fungus.
Results
In all four bioassays, female mosquitoes that had been exposed to conidia in the cylinder setup died significantly faster than the control females (F = 104.4; p < 0.001), with an average of 96.4 ± 2.0% of the cadavers having sporulating  M. anisopliae .M. anisopliaeAll three replicates of bioassay 1 indicated transfer of the fungus from contaminated females to uninfected males, with an average male infection rate of 26.1 ± 5.3% (Table  1 ). There was no difference in survival between the males of the control and test groups (F = 0.30; p = 0.5844), but when the group of test males was split into those that had been infected and those that had not, survival analysis showed a difference in survival approaching significance (F = 2.73; p = 0.098). Under a compound microscope, conidia were observed on four of the five males. Most were found on the lower parts (tibia, tarsi, uncinus (claws) and arolium) of the first and second pair of legs (Figure  1 ). A few conidia were found attached to the hairs of the tip of the wings. No conidia were found on the head, thorax, abdomen or the hind legs. Per mosquito 0–25 conidia were found. Female mosquitoes that had spent 24 hrs on fungus-impregnated paper had conidia on legs, tips of their wings and mouthparts, but not on the thorax or abdomen.Autodissemation of  M. anisopliae  from females to males  An. gambiae s.s .. The proportions of male mosquitoes infected are shown with the differences in survival of fungus-infected males compared to uninfected males within the test groups.M. anisopliaeAn. gambiae s.sa : SEM = standard error of meanb : Significant difference (p < 0.05) between infected and uninfected males using GLM analysis (Genstat 7.0).In the second bioassay, with individual pairs in glass vials, an average of 34.2 ± 0.6% of the males acquired and died of the fungal infection. Survival of these males as a group was not significantly different from the control group (F = 1.27; p = 0.259). When the males from the test group were split into those that became infected and those that did not, a decrease in survival of the infected test males compared to the uninfected males was observed, although this effect was not significant (F = 3.39; p = 0.065). However, the average age at death of 11.3 ± 1.9 days of the fungus-infected males was significantly lower than the 15.6 ± 1.6 days of the uninfected test males (p = 0.001).In the third bioassay, horizontal transmission occurred in nine of the 14 replicates (64.3%). In those nine trials an average of 16.7 ± 3.7% of the 10 males acquired an infection. Calculated over the 14 trials, the infection rate was 10.7 ± 3.2%. As in the other two bioassays, there was no significant difference in survival rates between the test and control groups (F = 2.19; p = 0.139). However, those males in the test group that were found with sporulating fungus died significantly faster than uninfected males (F = 13.02; p = 0.001) with the average age at the time of death of 8.4 ± 1.1 days for infected and 17.9 ± 0.6 days for uninfected males, respectively.None of the 114 males in bioassay 4 were found infected with the fungus.Discussion It is generally believed that fungal dissemination within a host population occurs due to activities and movements of the host. The fungus can exploit host behaviour like trophallaxis, tactile communication, grooming (in social insects) [ 29 , 30 ] and mating [ 31 ] to spread through a host population. Taking into account the physiological state of the females and the natural display of behaviour at the time of the bioassays, it is assumed that the observed autodissemination of  M. anisopliae  from female to male  An. gambiae s.s . was the result of mating. This is strongly supported by the findings from experiment 4 where none of the males that had stayed on the surface area where fungus-contaminated females had rested previously acquired an infection. The average age at death of fungus-infected mosquitoes was quite high when compared to mosquito survival in Scholte  et al . [ 14 ]. This is probably due to the relatively low level (a maximum of 25 conidia) of inoculum transferred. From those mosquitoes that were checked under the microscope for the presence of conidia, four out of five males contained conidia. It is thus likely that many males become contaminated, but that only a relatively low proportion of these males will actually succumb to the infection: in many cases the number of conidia was low, resulting in marginal infections that were successfully countered by the immune responses staged by the males. In order to achieve the highest possible impact of the fungus on the mosquito population, it is desirable that other pathways besides the primary mode of (direct) contamination are utilized. The results of this study show that under laboratory conditions horizontal transmission can occur, which suggests that it may occur in the field. When these experiments were carried out, it was presumed that predominantly females would be infected directly from the indoor resting targets in the field. From a recent field experiment (Scholte  et al ., in preparation), however, it was found that a large proportion (44.9%) of the  An. gambiae s.l . found indoors were males. This suggests that not only females can deliver fungal inoculum to uninfected males, but that also infected males may infect uninfected females. Further research is needed to determine to what extent this secondary pathway of fungal contamination may contribute to spreading the fungus within mosquito field populations.
Discussion
It is generally believed that fungal dissemination within a host population occurs due to activities and movements of the host. The fungus can exploit host behaviour like trophallaxis, tactile communication, grooming (in social insects) [ 29 , 30 ] and mating [ 31 ] to spread through a host population. Taking into account the physiological state of the females and the natural display of behaviour at the time of the bioassays, it is assumed that the observed autodissemination of  M. anisopliae  from female to male  An. gambiae s.s . was the result of mating. This is strongly supported by the findings from experiment 4 where none of the males that had stayed on the surface area where fungus-contaminated females had rested previously acquired an infection. The average age at death of fungus-infected mosquitoes was quite high when compared to mosquito survival in Scholte  et al . [ 14 ]. This is probably due to the relatively low level (a maximum of 25 conidia) of inoculum transferred. From those mosquitoes that were checked under the microscope for the presence of conidia, four out of five males contained conidia. It is thus likely that many males become contaminated, but that only a relatively low proportion of these males will actually succumb to the infection: in many cases the number of conidia was low, resulting in marginal infections that were successfully countered by the immune responses staged by the males.M. anisopliaeAn. gambiae s.set alIn order to achieve the highest possible impact of the fungus on the mosquito population, it is desirable that other pathways besides the primary mode of (direct) contamination are utilized. The results of this study show that under laboratory conditions horizontal transmission can occur, which suggests that it may occur in the field. When these experiments were carried out, it was presumed that predominantly females would be infected directly from the indoor resting targets in the field. From a recent field experiment (Scholte  et al ., in preparation), however, it was found that a large proportion (44.9%) of the  An. gambiae s.l . found indoors were males. This suggests that not only females can deliver fungal inoculum to uninfected males, but that also infected males may infect uninfected females. Further research is needed to determine to what extent this secondary pathway of fungal contamination may contribute to spreading the fungus within mosquito field populations.et alAn. gambiae s.lConclusion This study has shown that horizontal transfer of fungal inoculum between mosquitoes is possible during copulation and may contribute to spreading of the fungus within target mosquito populations in the field. However, since conditions under which horizontal transmission is likely to occur are quite specific, field experimentation is required to measure the real impact that autodissemination may have. For now it is concluded that the relatively low infection levels recorded in this study suggest that the impact of biological control with  M. anisopliae  against African anophelines will predominantly depend on direct contamination of adult mosquitoes from conidia-impregnated resting targets such as walls, ceilings and sheets.
Conclusion
This study has shown that horizontal transfer of fungal inoculum between mosquitoes is possible during copulation and may contribute to spreading of the fungus within target mosquito populations in the field. However, since conditions under which horizontal transmission is likely to occur are quite specific, field experimentation is required to measure the real impact that autodissemination may have. For now it is concluded that the relatively low infection levels recorded in this study suggest that the impact of biological control with  M. anisopliae  against African anophelines will predominantly depend on direct contamination of adult mosquitoes from conidia-impregnated resting targets such as walls, ceilings and sheets.M. anisopliaeAuthors' contribution E-JS was directly involved in the experimental work, analysis of the data and drafting of the manuscript. BGJK conceived of the study, obtained funding for it in collaboration with WT and revised the manuscript prior to submission.
Authors' contribution
E-JS was directly involved in the experimental work, analysis of the data and drafting of the manuscript. BGJK conceived of the study, obtained funding for it in collaboration with WT and revised the manuscript prior to submission.


Background Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of arterial aneurysms through increased proteolysis of extracellular matrix proteins. Increased proteolysis due to elevated matrix degrading enzyme activity in the arterial wall may act as a susceptibility factor for the development of coronary aneurysms. The aim of this study was to investigate the association between MMPs and presence of coronary aneurysms.
Background
Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of arterial aneurysms through increased proteolysis of extracellular matrix proteins. Increased proteolysis due to elevated matrix degrading enzyme activity in the arterial wall may act as a susceptibility factor for the development of coronary aneurysms. The aim of this study was to investigate the association between MMPs and presence of coronary aneurysms.Methods Thirty patients with aneurysmal coronary artery disease and stable angina were enrolled into study (Group 1). Fourteen coronary artery disease patients with stable angina were selected as control group (Group 2). MMP-1, MMP-3 and C-reactive protein (CRP) were measured in peripheral venous blood and matched between the groups.
Methods
Thirty patients with aneurysmal coronary artery disease and stable angina were enrolled into study (Group 1). Fourteen coronary artery disease patients with stable angina were selected as control group (Group 2). MMP-1, MMP-3 and C-reactive protein (CRP) were measured in peripheral venous blood and matched between the groups.Results Serum MMP-3 level was higher in patients with aneurismal coronary artery disease compared to the control group (20.23 ± 14.68 vs 11.45 ± 6.55 ng/ml, p = 0.039). Serum MMP-1 (13.63 ± 7.73 vs 12.15 ± 6.27 ng/ml, p = 0.52) and CRP levels (4.78 ± 1.47 vs 4.05 ± 1.53 mg/l, p = 0.13) were not significantly different between the groups.
Results
Serum MMP-3 level was higher in patients with aneurismal coronary artery disease compared to the control group (20.23 ± 14.68 vs 11.45 ± 6.55 ng/ml, p = 0.039). Serum MMP-1 (13.63 ± 7.73 vs 12.15 ± 6.27 ng/ml, p = 0.52) and CRP levels (4.78 ± 1.47 vs 4.05 ± 1.53 mg/l, p = 0.13) were not significantly different between the groups.Conclusion MMPs can cause arterial wall destruction. MMP-3 may play role in the pathogenesis of coronary aneurysm development through increased proteolysis of extracellular matrix proteins.
Conclusion
MMPs can cause arterial wall destruction. MMP-3 may play role in the pathogenesis of coronary aneurysm development through increased proteolysis of extracellular matrix proteins.

Introduction Coronary artery aneurysms are defined as dilated coronary artery segments that are greater than 1.5 times the diameter of adjacent normal segments [ 1 , 2 ]. The gold standard for diagnosing this type of aneurysm is coronary angiography, which provides information about the size, shape, location and number of aneurysms. Coronary aneurysms may occur during the development of coronary atherosclerosis. Previous studies have shown that coronary aneurysms are observed in 1% to 5% of patients with angiographic evidence of coronary artery disease [ 3 - 6 ]. In some studies, coronary aneurysms have been associated with an increased risk of myocardial infarction [ 3 , 4 ]. Although the mechanisms responsible for coronary aneurysm formation during the atherosclerotic process are unclear, atherosclerosis-induced aneurysms derive primarily from thinning and/or destruction of the media [ 6 - 8 ]. Possible factors contributing to aneurysms are matrix-degrading enzymes such as collagenases, gelatinases, and stromelysins [ 9 , 10 ]. More specifically, matrix metalloproteinases (MMPs) are enzymes that can degrade the structural proteins of connective tissue. Degradation of extracellular matrix proteins may weaken the connective tissue, thereby leading to a weakened vascular wall. We investigated the association between MMPs and coronary artery aneurysm by measuring the levels of MMP-1 and MMP-3 (both of which represent markers of proteolytic activity) in patients with coronary artery disease, some of whom had coronary aneurysms (cases) and others who did not (controls).
Introduction
Coronary artery aneurysms are defined as dilated coronary artery segments that are greater than 1.5 times the diameter of adjacent normal segments [ 1 , 2 ]. The gold standard for diagnosing this type of aneurysm is coronary angiography, which provides information about the size, shape, location and number of aneurysms.Coronary aneurysms may occur during the development of coronary atherosclerosis. Previous studies have shown that coronary aneurysms are observed in 1% to 5% of patients with angiographic evidence of coronary artery disease [ 3 - 6 ]. In some studies, coronary aneurysms have been associated with an increased risk of myocardial infarction [ 3 , 4 ]. Although the mechanisms responsible for coronary aneurysm formation during the atherosclerotic process are unclear, atherosclerosis-induced aneurysms derive primarily from thinning and/or destruction of the media [ 6 - 8 ].Possible factors contributing to aneurysms are matrix-degrading enzymes such as collagenases, gelatinases, and stromelysins [ 9 , 10 ]. More specifically, matrix metalloproteinases (MMPs) are enzymes that can degrade the structural proteins of connective tissue. Degradation of extracellular matrix proteins may weaken the connective tissue, thereby leading to a weakened vascular wall.We investigated the association between MMPs and coronary artery aneurysm by measuring the levels of MMP-1 and MMP-3 (both of which represent markers of proteolytic activity) in patients with coronary artery disease, some of whom had coronary aneurysms (cases) and others who did not (controls).Methods Patient population We reviewed the medical records of patients who had undergone coronary angiography between January, 2002 and April, 2003. Among 4,456 cases reviewed, 55 patients (1.23%) diagnosed with aneurysmal coronary artery disease were selected. Sixteen patients with acute coronary syndromes and nine patients with balloon angioplasty history were excluded from the study. The remaining 30 patients with aneurysmal coronary artery disease patients were enrolled into the study. Transverse diameter of an aneurysm and reference vessel were measured using the post-processing software (Schimadzu Corporation, DIGITEX ALPHA Plus System, Kyoto, Japan, 2001). The ratio between dilated coronary artery segment and reference vessel diameter was calculated. The control patients (n = 14) had coronary artery disease, but were free of aneurysmal coronary dilatation. Both groups had positive exercise stress tests and had been diagnosed with stable angina. Blood biochemistry and echocardiography were performed in all patients. No patient had a history of coronary atherectomy or balloon angioplasty. All participants gave informed consent. Autoimmune disease, inflammatory arteritis, chronic or, acute infectious disease, use of steroid or anti-inflammatory drugs within the last three months, renal failure and cancer were accepted as exclusion criteria. Laboratory assays Specimen collection Fasting blood samples (8–10 hours fast) were obtained from the antecubital vein at approximately 9:00 a.m. These were centrifuged for 10 min at 3,000 × g at a temperature of about 4°C. Serum was stored at -70°C. Blood samples were analyzed at the Ege University Department of Microbiology, Section of Serology. Assay protocol for MMP-1 and MMP-3 MMP levels were determined using enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer's instructions (MMP-1, Biotrak Amersham Pharmacia Biotech, United Kingdom; RPN 2610; MMP-3, Biotrak Amersham Pharmacia Biotech, United Kingdom; RPN 2613). The ELISA kit measured total MMP-1 (pro MMP-1, free MMP-1, MMP1/tissue inhibitor MP-1 complex), total MMP-3 (pro MMP-3, free MMP-3, MMP3/tissue inhibitor MP-1 and MMP3/tissue inhibitor MP-2 complex) at >89% cross reactivity. Samples were incubated in microtitre wells pre-coated with anti-MMP-1 (lyophilized rabbit anti-MMP-1) and anti-MMP-3 (peroxidase labelled Fab antibody to MMP-3) antibodies. The assays use the pro form of a detection enzyme that can be activated (by captured active MMP) into an active detection enzyme. MMP-1 and MMP-3 can be measured in the range of 6.25–100 ng/ml and 3.75–120 ng/ml, respectively. The results received from the optic scanners at 450 nm were converted into ng/ml values from a standard curve. All samples were run in duplicate and were averaged. Within-assay precision values for duplicate determinations were 5.5%, 7.9% and 7.3% at MMP-1 concentrations of 16.89 ± 0.94 ng/ml, 35.53 ± 2.82 ng/ml and 54.08 ± 4.0 ng/ml, respectively. Between-assay precisions for repeated measurements of the same sample were 11.6%, 12.0% and 13.2% at MMP-1 concentrations of 23.19 ± 2.68 ng/ml, 55.27 ± 6.65 and 98.04 ± 12.93, respectively. The within-assay precisions for duplicate determinations were 4.8%, 2.4% and 2.1% at MMP-3 concentrations of 13.7 ± 0.66 ng/ml, 33.7 ± 0.83 ng/ml and 83.2 ± 1.76 ng/ml, respectively. Between-assay precisions for repeated measurement of the same sample were 13.3%, 11.7% and 8.8% at MMP-3 concentrations of 11.2 ± 1.49 ng/ml, 27.6 ± 3.24 and 75.4 ± 6.63, respectively. Determination of C-reactive protein levels Serums were obtained by centrifugation of vacutainer-clotted tubes at 3,000 rpm for 10 minutes. High sensitivity C-reactive protein (hs-CRP) samples were stored at -30°C and analyzed by latex particle-enhanced immunoturbidimetric assay. The total median inter-assay and intra-assay coefficients of variation for the assays were <6% for CRP. All results were recorded in the patients' files. Statistical analyses All values are reported as mean ± SD. Chi Square test was used in the comparison of categorical variables while student unpaired-t test or Mann-Whitney Rank Sum tests were used, where appropriate, in the univariate analysis. Statistical analyses were performed with SPSS statistical software. A value of p < 0.05 was considered to be statistically significant.
Methods
Patient population We reviewed the medical records of patients who had undergone coronary angiography between January, 2002 and April, 2003. Among 4,456 cases reviewed, 55 patients (1.23%) diagnosed with aneurysmal coronary artery disease were selected. Sixteen patients with acute coronary syndromes and nine patients with balloon angioplasty history were excluded from the study. The remaining 30 patients with aneurysmal coronary artery disease patients were enrolled into the study. Transverse diameter of an aneurysm and reference vessel were measured using the post-processing software (Schimadzu Corporation, DIGITEX ALPHA Plus System, Kyoto, Japan, 2001). The ratio between dilated coronary artery segment and reference vessel diameter was calculated. The control patients (n = 14) had coronary artery disease, but were free of aneurysmal coronary dilatation. Both groups had positive exercise stress tests and had been diagnosed with stable angina. Blood biochemistry and echocardiography were performed in all patients. No patient had a history of coronary atherectomy or balloon angioplasty. All participants gave informed consent. Autoimmune disease, inflammatory arteritis, chronic or, acute infectious disease, use of steroid or anti-inflammatory drugs within the last three months, renal failure and cancer were accepted as exclusion criteria.
Patient population
We reviewed the medical records of patients who had undergone coronary angiography between January, 2002 and April, 2003. Among 4,456 cases reviewed, 55 patients (1.23%) diagnosed with aneurysmal coronary artery disease were selected. Sixteen patients with acute coronary syndromes and nine patients with balloon angioplasty history were excluded from the study. The remaining 30 patients with aneurysmal coronary artery disease patients were enrolled into the study. Transverse diameter of an aneurysm and reference vessel were measured using the post-processing software (Schimadzu Corporation, DIGITEX ALPHA Plus System, Kyoto, Japan, 2001). The ratio between dilated coronary artery segment and reference vessel diameter was calculated. The control patients (n = 14) had coronary artery disease, but were free of aneurysmal coronary dilatation. Both groups had positive exercise stress tests and had been diagnosed with stable angina. Blood biochemistry and echocardiography were performed in all patients. No patient had a history of coronary atherectomy or balloon angioplasty. All participants gave informed consent.Autoimmune disease, inflammatory arteritis, chronic or, acute infectious disease, use of steroid or anti-inflammatory drugs within the last three months, renal failure and cancer were accepted as exclusion criteria.Laboratory assays Specimen collection Fasting blood samples (8–10 hours fast) were obtained from the antecubital vein at approximately 9:00 a.m. These were centrifuged for 10 min at 3,000 × g at a temperature of about 4°C. Serum was stored at -70°C. Blood samples were analyzed at the Ege University Department of Microbiology, Section of Serology. Assay protocol for MMP-1 and MMP-3 MMP levels were determined using enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer's instructions (MMP-1, Biotrak Amersham Pharmacia Biotech, United Kingdom; RPN 2610; MMP-3, Biotrak Amersham Pharmacia Biotech, United Kingdom; RPN 2613). The ELISA kit measured total MMP-1 (pro MMP-1, free MMP-1, MMP1/tissue inhibitor MP-1 complex), total MMP-3 (pro MMP-3, free MMP-3, MMP3/tissue inhibitor MP-1 and MMP3/tissue inhibitor MP-2 complex) at >89% cross reactivity. Samples were incubated in microtitre wells pre-coated with anti-MMP-1 (lyophilized rabbit anti-MMP-1) and anti-MMP-3 (peroxidase labelled Fab antibody to MMP-3) antibodies. The assays use the pro form of a detection enzyme that can be activated (by captured active MMP) into an active detection enzyme. MMP-1 and MMP-3 can be measured in the range of 6.25–100 ng/ml and 3.75–120 ng/ml, respectively. The results received from the optic scanners at 450 nm were converted into ng/ml values from a standard curve. All samples were run in duplicate and were averaged. Within-assay precision values for duplicate determinations were 5.5%, 7.9% and 7.3% at MMP-1 concentrations of 16.89 ± 0.94 ng/ml, 35.53 ± 2.82 ng/ml and 54.08 ± 4.0 ng/ml, respectively. Between-assay precisions for repeated measurements of the same sample were 11.6%, 12.0% and 13.2% at MMP-1 concentrations of 23.19 ± 2.68 ng/ml, 55.27 ± 6.65 and 98.04 ± 12.93, respectively. The within-assay precisions for duplicate determinations were 4.8%, 2.4% and 2.1% at MMP-3 concentrations of 13.7 ± 0.66 ng/ml, 33.7 ± 0.83 ng/ml and 83.2 ± 1.76 ng/ml, respectively. Between-assay precisions for repeated measurement of the same sample were 13.3%, 11.7% and 8.8% at MMP-3 concentrations of 11.2 ± 1.49 ng/ml, 27.6 ± 3.24 and 75.4 ± 6.63, respectively. Determination of C-reactive protein levels Serums were obtained by centrifugation of vacutainer-clotted tubes at 3,000 rpm for 10 minutes. High sensitivity C-reactive protein (hs-CRP) samples were stored at -30°C and analyzed by latex particle-enhanced immunoturbidimetric assay. The total median inter-assay and intra-assay coefficients of variation for the assays were <6% for CRP. All results were recorded in the patients' files.
Laboratory assays
Specimen collection Fasting blood samples (8–10 hours fast) were obtained from the antecubital vein at approximately 9:00 a.m. These were centrifuged for 10 min at 3,000 × g at a temperature of about 4°C. Serum was stored at -70°C. Blood samples were analyzed at the Ege University Department of Microbiology, Section of Serology.
Specimen collection
Fasting blood samples (8–10 hours fast) were obtained from the antecubital vein at approximately 9:00 a.m. These were centrifuged for 10 min at 3,000 × g at a temperature of about 4°C. Serum was stored at -70°C. Blood samples were analyzed at the Ege University Department of Microbiology, Section of Serology.Assay protocol for MMP-1 and MMP-3 MMP levels were determined using enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer's instructions (MMP-1, Biotrak Amersham Pharmacia Biotech, United Kingdom; RPN 2610; MMP-3, Biotrak Amersham Pharmacia Biotech, United Kingdom; RPN 2613). The ELISA kit measured total MMP-1 (pro MMP-1, free MMP-1, MMP1/tissue inhibitor MP-1 complex), total MMP-3 (pro MMP-3, free MMP-3, MMP3/tissue inhibitor MP-1 and MMP3/tissue inhibitor MP-2 complex) at >89% cross reactivity. Samples were incubated in microtitre wells pre-coated with anti-MMP-1 (lyophilized rabbit anti-MMP-1) and anti-MMP-3 (peroxidase labelled Fab antibody to MMP-3) antibodies. The assays use the pro form of a detection enzyme that can be activated (by captured active MMP) into an active detection enzyme. MMP-1 and MMP-3 can be measured in the range of 6.25–100 ng/ml and 3.75–120 ng/ml, respectively. The results received from the optic scanners at 450 nm were converted into ng/ml values from a standard curve. All samples were run in duplicate and were averaged. Within-assay precision values for duplicate determinations were 5.5%, 7.9% and 7.3% at MMP-1 concentrations of 16.89 ± 0.94 ng/ml, 35.53 ± 2.82 ng/ml and 54.08 ± 4.0 ng/ml, respectively. Between-assay precisions for repeated measurements of the same sample were 11.6%, 12.0% and 13.2% at MMP-1 concentrations of 23.19 ± 2.68 ng/ml, 55.27 ± 6.65 and 98.04 ± 12.93, respectively. The within-assay precisions for duplicate determinations were 4.8%, 2.4% and 2.1% at MMP-3 concentrations of 13.7 ± 0.66 ng/ml, 33.7 ± 0.83 ng/ml and 83.2 ± 1.76 ng/ml, respectively. Between-assay precisions for repeated measurement of the same sample were 13.3%, 11.7% and 8.8% at MMP-3 concentrations of 11.2 ± 1.49 ng/ml, 27.6 ± 3.24 and 75.4 ± 6.63, respectively.
Assay protocol for MMP-1 and MMP-3
MMP levels were determined using enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer's instructions (MMP-1, Biotrak Amersham Pharmacia Biotech, United Kingdom; RPN 2610; MMP-3, Biotrak Amersham Pharmacia Biotech, United Kingdom; RPN 2613). The ELISA kit measured total MMP-1 (pro MMP-1, free MMP-1, MMP1/tissue inhibitor MP-1 complex), total MMP-3 (pro MMP-3, free MMP-3, MMP3/tissue inhibitor MP-1 and MMP3/tissue inhibitor MP-2 complex) at >89% cross reactivity. Samples were incubated in microtitre wells pre-coated with anti-MMP-1 (lyophilized rabbit anti-MMP-1) and anti-MMP-3 (peroxidase labelled Fab antibody to MMP-3) antibodies. The assays use the pro form of a detection enzyme that can be activated (by captured active MMP) into an active detection enzyme. MMP-1 and MMP-3 can be measured in the range of 6.25–100 ng/ml and 3.75–120 ng/ml, respectively. The results received from the optic scanners at 450 nm were converted into ng/ml values from a standard curve. All samples were run in duplicate and were averaged. Within-assay precision values for duplicate determinations were 5.5%, 7.9% and 7.3% at MMP-1 concentrations of 16.89 ± 0.94 ng/ml, 35.53 ± 2.82 ng/ml and 54.08 ± 4.0 ng/ml, respectively. Between-assay precisions for repeated measurements of the same sample were 11.6%, 12.0% and 13.2% at MMP-1 concentrations of 23.19 ± 2.68 ng/ml, 55.27 ± 6.65 and 98.04 ± 12.93, respectively. The within-assay precisions for duplicate determinations were 4.8%, 2.4% and 2.1% at MMP-3 concentrations of 13.7 ± 0.66 ng/ml, 33.7 ± 0.83 ng/ml and 83.2 ± 1.76 ng/ml, respectively. Between-assay precisions for repeated measurement of the same sample were 13.3%, 11.7% and 8.8% at MMP-3 concentrations of 11.2 ± 1.49 ng/ml, 27.6 ± 3.24 and 75.4 ± 6.63, respectively.Determination of C-reactive protein levels Serums were obtained by centrifugation of vacutainer-clotted tubes at 3,000 rpm for 10 minutes. High sensitivity C-reactive protein (hs-CRP) samples were stored at -30°C and analyzed by latex particle-enhanced immunoturbidimetric assay. The total median inter-assay and intra-assay coefficients of variation for the assays were <6% for CRP. All results were recorded in the patients' files.
Determination of C-reactive protein levels
Serums were obtained by centrifugation of vacutainer-clotted tubes at 3,000 rpm for 10 minutes. High sensitivity C-reactive protein (hs-CRP) samples were stored at -30°C and analyzed by latex particle-enhanced immunoturbidimetric assay. The total median inter-assay and intra-assay coefficients of variation for the assays were <6% for CRP. All results were recorded in the patients' files.Statistical analyses All values are reported as mean ± SD. Chi Square test was used in the comparison of categorical variables while student unpaired-t test or Mann-Whitney Rank Sum tests were used, where appropriate, in the univariate analysis. Statistical analyses were performed with SPSS statistical software. A value of p < 0.05 was considered to be statistically significant.
Statistical analyses
All values are reported as mean ± SD. Chi Square test was used in the comparison of categorical variables while student unpaired-t test or Mann-Whitney Rank Sum tests were used, where appropriate, in the univariate analysis. Statistical analyses were performed with SPSS statistical software. A value of p < 0.05 was considered to be statistically significant.Results There were no significant differences in baseline characteristics between cases and controls. High-density lipoprotein, low-density lipoprotein, total cholesterol and triglyceride levels were not statistically different between the groups. Clinical characteristics of and medication use by the groups are shown in Table  1 . Table 1 Clinical Characteristics and Medication Use of Study Participants Group 1 (n = 30) Group 2 (n = 14) p Mean age (yrs) 55.2 ± 10.0 51.8 ± 7.7 NS Male sex % (n) 70%(21) 64%(9) NS Diabetes Mellitus % (n) 13%(4) 14%(2) NS Hypertension % (n) 30%(9) 21%(3) NS Smoking % (n) 60%(18) 50%(7) NS TC (mg/dl) 196.8 ± 31.7 195.1 ± 38.2 NS TG (mg/dl) 148.7 ± 71.2 151.7 ± 64.0 NS HDL-C (mg/dl) 46.7 ± 11.8 50.7 ± 13.0 NS LDL-C (mg/dl) 125.1 ± 28.2 116.7 ± 34.2 NS hs-CRP (mg/L) 4.78 ± 1.47 4.05 ± 1.53 NS MMP-1 (ng/ml) 13.63 ± 7.73 12.15 ± 6.27 NS MMP-3 (ng/ml) 20.23 ± 14.68 11.45 ± 6.55 0.039 Baseline therapy Aspirin 73%(22) 64%(9) NS Nitrate 57%(17) 64%(9) NS Statin 17%(5) 21%(3) NS Number of stenotic vessels One vessel disease 37%(11) 36%(5) NS Two vessel disease 50%(15) 43%(6) NS Three vessel disease 13%(4) 21%(3) NS Reference vessel diameter (mm) 2.95 ± 0.48 - - Aneurysm vessel diameter (mm) 4.78 ± 0.93 - - Aneurysm/reference vessel ratio 1.6 ± 0.1 - - Aneurysm segment Right coronary artery 53%(16) - - Left anterior descending artery 27%(8) - - Left Circumflex artery 30%(9) - - Group 1: Patients with coronary aneurysm,  Group 2: Patients without coronary aneurysm,  TC: Total cholesterol,  TG: Triglyceride,  HDL-C: High-density lipoprotein cholesterol,  LDL-C: Low-density lipoprotein cholesterol,  hs-CRP: High sensitivity C- reactive protein,  MMP-1: Matrix metalloproteinase-1,  MMP-3: Matrix metalloproteinase-3,  NS: Non-significant Mean serum MMP-1 (13.63 ± 7.73 vs 12.15 ± 6.27 ng/ml, p = 0.52) and CRP levels (4.78 ± 1.47 vs 4.05 ± 1.53 mg/l, p = 0.13) were not significantly different between cases and controls. Mean serum MMP-3 values were significantly higher in the cases than in controls (20.23 ± 14.68 and 11.45 ± 6.55 ng/ml respectively, p = 0.039). MMP-1, MMP-3 and hs-CRP levels are shown in Figure  1 . Figure 1 text
Results
There were no significant differences in baseline characteristics between cases and controls. High-density lipoprotein, low-density lipoprotein, total cholesterol and triglyceride levels were not statistically different between the groups. Clinical characteristics of and medication use by the groups are shown in Table  1 .Clinical Characteristics and Medication Use of Study ParticipantsGroup 1 (n = 30)Group 2 (n = 14)pMean age (yrs)Male sex % (n)Diabetes Mellitus % (n)Hypertension % (n)Smoking % (n)TC (mg/dl)TG (mg/dl)HDL-C (mg/dl)LDL-C (mg/dl)hs-CRP (mg/L)MMP-1 (ng/ml)MMP-3 (ng/ml)Baseline therapyNumber of stenotic vesselsReference vessel diameter (mm)Aneurysm vessel diameter (mm)Aneurysm/reference vessel ratioAneurysm segmentGroup 1: Patients with coronary aneurysm,  Group 2: Patients without coronary aneurysm,  TC: Total cholesterol,  TG: Triglyceride,  HDL-C: High-density lipoprotein cholesterol,  LDL-C: Low-density lipoprotein cholesterol,  hs-CRP: High sensitivity C- reactive protein,  MMP-1: Matrix metalloproteinase-1,  MMP-3: Matrix metalloproteinase-3,  NS: Non-significantGroup 1:Group 2:TC:TG:HDL-C:LDL-C:hs-CRP:MMP-1:MMP-3:NS:Mean serum MMP-1 (13.63 ± 7.73 vs 12.15 ± 6.27 ng/ml, p = 0.52) and CRP levels (4.78 ± 1.47 vs 4.05 ± 1.53 mg/l, p = 0.13) were not significantly different between cases and controls. Mean serum MMP-3 values were significantly higher in the cases than in controls (20.23 ± 14.68 and 11.45 ± 6.55 ng/ml respectively, p = 0.039). MMP-1, MMP-3 and hs-CRP levels are shown in Figure  1 .Discussion Essential factors contributing to the formation of coronary aneurysms include vessel media degradation and ulceration due to increased proteolytic activity. Connective tissue integrity, another factor contributing to aneurysm development, depends on the balance between degradation and repair of the extracellular matrix. Activation or inhibition of degrading enzymes affects extracellular matrix modeling [ 9 , 10 ], which, in turn, affects connective tissue and vascular wall integrity. Matrix-degrading enzyme activity is a tightly controlled process that involves transcription, activation of latent pro-enzymes and inhibition of proteolytic activity [ 11 - 13 ]. A key step in the regulation of MMPs may occur at the level of transcription [ 14 ]. The mechanism by which gene transcription is mediated is thought to involve a prostaglandin E 2 (PGE 2 )-cAMP- dependent pathway. G-proteins have been implicated in this pathway [ 15 ]. Transcription activity can be stimulated by a variety of inflammatory cytokines, hormones, and growth factors [ 16 - 19 ]. Several factors are also known to inhibit MMP gene expression and these include indomethacin, corticosteroids, and interleukin-4 [ 17 , 20 , 21 ]. MMP activity is also regulated by tissue-specific inhibitors. There are four known tissue inhibitors of metalloproteinases (TIMP-1, -2, -3 and -4). The TIMPs are secreted by a variety of cell lines, including smooth muscle cells and macrophages. Their activity is increased by growth factors and either increased or decreased by different interleukins [ 22 ]. Increased levels of MMP-2, MMP-3, MMP-9 and MMP-12 have been identified in aneurysm vessel walls [ 23 - 27 ]. Gene disruption of MMP-9 suppresses the development of experimental abdominal aortic aneurysms [ 28 ]. Conversely, decreased levels of TIMPs have been found in the aneurysm wall [ 26 ]. Allaire et al. [ 29 ] reported that local expression of TIMP-1 may prevent aortic aneurysm degeneration and rupture in a rat model. Carrell et al. [ 30 ] examined differences in MMPs between patients with aortic aneurysm and patients with aortic atherosclerosis but without aneurysm. Among a wide range of MMPs tested, only MMP-3 was over-expressed in the aortic aneurysm samples. Reduced aneurysm formation has been observed in mice with MMP-3 gene inactivation [ 31 ]. Finally, the recent observation that high circulating levels of MMP-3 are associated with coronary lesions in Kawasaki disease [ 32 ] also supports an important role for MMP-3 in the pathogenesis of coronary aneurysms. These data suggest that proteolytic balance in the vascular wall plays a key role in aneurysm development. MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) are members of a family of proteinases that degrade one or more components of the extracellular matrix. In our study, it appears that elevated MMP-3 activity may represent a risk factor for coronary aneurysm formation. This finding is concordant with previously published studies. The mechanisms underlying this association are unclear. MMP-3 gene disruption may be responsible. Lamblin et al. [ 33 ] have reported similar findings, namely, that the MMP-3 5A allele is associated with the occurrence of coronary aneurysm. Others have reported that MMP-3 is expressed in atherosclerotic plaque cells, but not by cells in normal arteries [ 34 - 37 ]. In addition, extensive inflammation and destruction of musculo-elastic vessel wall elements have been observed in dilated human coronary arteries [ 38 , 39 ]. Schoenhagen et al. [ 40 ] suggest that the degradation of extracellular matrix by MMP-3 may contribute to the expansion of the coronary vessel wall. This effect is characteristic of positive remodeling. Based on these and our own observations, we maintain that MMP-3 over-expression may occur in aneurysm segments. Histopathologic studies would be needed to clarify whether or not this is the case. MMP levels are elevated in patients with acute myocardial infarction, unstable angina and coronary angioplasty [ 35 , 41 , 42 ]. All patients in our study had been diagnosed with stable angina before being enrolled into the study. CRP reflects systemic inflammatory activity. In this study, we did not observe increased CRP levels in those patients with coronary aneurysms. One explanation for similar CRP expression between cases and controls might be that all study subjects had been diagnosed with stable angina pectoris. Varying degrees of inflammation are reported among individuals with abdominal aortic aneurysms. This variation may relate to possible confounding due to clinical manifestations (asymptomatic or symptomatic) and aneurysm progression rates (cm/year). Other investigators have failed to observe increased CRP levels among asymptomatic patients with abdominal aortic aneurysm [ 43 ]. Because elevated MMP-3 levels likely contribute to the development of coronary aneurysms, this matrix-degrading enzyme may represent an important therapeutic target. Luan et al. [ 44 ] reported that a number of statins inhibit MMP-3 activity in rabbits. COX-2 inhibitors may also suppress MMP expression. Production of MMPs by macrophages occurs through a PGE 2 /cAMP-dependent pathway [ 45 ]. Theoretically, COX-2 inhibitors could attenuate this pathway. Another target of MMP inhibition has been demonstrated in animal models of adenovirus-mediated TIMP gene transfer [ 46 ]. In reporting our findings, we acknowledge that measurement of TIMP levels between cases and controls would have provided useful information about the possibility of proteolytic imbalance. Similarly, measurement of locally produced inflammatory cytokines, hormones and growth factors would be interesting to know about, since these regulate matrix-degrading enzyme expression [ 16 - 19 ]. This could provide relevant information, as systemic inflammatory activity may not reflect local inflammatory infiltration in aneurysm segments. Finally, the study would have benefited from having a larger sample size as well as genotype determination. We conclude that MMP-3 overexpression due to a proteolytic imbalance may lead to coronary aneurysm development through degradation of matrix components, especially lamina elastica. New medical therapeutic options targeted specifically against MMP-3 may prove useful in the prevention of aneurysm formation.
Discussion
Essential factors contributing to the formation of coronary aneurysms include vessel media degradation and ulceration due to increased proteolytic activity. Connective tissue integrity, another factor contributing to aneurysm development, depends on the balance between degradation and repair of the extracellular matrix. Activation or inhibition of degrading enzymes affects extracellular matrix modeling [ 9 , 10 ], which, in turn, affects connective tissue and vascular wall integrity.Matrix-degrading enzyme activity is a tightly controlled process that involves transcription, activation of latent pro-enzymes and inhibition of proteolytic activity [ 11 - 13 ]. A key step in the regulation of MMPs may occur at the level of transcription [ 14 ]. The mechanism by which gene transcription is mediated is thought to involve a prostaglandin E 2 (PGE 2 )-cAMP- dependent pathway. G-proteins have been implicated in this pathway [ 15 ]. Transcription activity can be stimulated by a variety of inflammatory cytokines, hormones, and growth factors [ 16 - 19 ]. Several factors are also known to inhibit MMP gene expression and these include indomethacin, corticosteroids, and interleukin-4 [ 17 , 20 , 21 ].MMP activity is also regulated by tissue-specific inhibitors. There are four known tissue inhibitors of metalloproteinases (TIMP-1, -2, -3 and -4). The TIMPs are secreted by a variety of cell lines, including smooth muscle cells and macrophages. Their activity is increased by growth factors and either increased or decreased by different interleukins [ 22 ]. Increased levels of MMP-2, MMP-3, MMP-9 and MMP-12 have been identified in aneurysm vessel walls [ 23 - 27 ]. Gene disruption of MMP-9 suppresses the development of experimental abdominal aortic aneurysms [ 28 ]. Conversely, decreased levels of TIMPs have been found in the aneurysm wall [ 26 ]. Allaire et al. [ 29 ] reported that local expression of TIMP-1 may prevent aortic aneurysm degeneration and rupture in a rat model. Carrell et al. [ 30 ] examined differences in MMPs between patients with aortic aneurysm and patients with aortic atherosclerosis but without aneurysm. Among a wide range of MMPs tested, only MMP-3 was over-expressed in the aortic aneurysm samples. Reduced aneurysm formation has been observed in mice with MMP-3 gene inactivation [ 31 ]. Finally, the recent observation that high circulating levels of MMP-3 are associated with coronary lesions in Kawasaki disease [ 32 ] also supports an important role for MMP-3 in the pathogenesis of coronary aneurysms. These data suggest that proteolytic balance in the vascular wall plays a key role in aneurysm development.MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) are members of a family of proteinases that degrade one or more components of the extracellular matrix. In our study, it appears that elevated MMP-3 activity may represent a risk factor for coronary aneurysm formation. This finding is concordant with previously published studies. The mechanisms underlying this association are unclear. MMP-3 gene disruption may be responsible. Lamblin et al. [ 33 ] have reported similar findings, namely, that the MMP-3 5A allele is associated with the occurrence of coronary aneurysm.Others have reported that MMP-3 is expressed in atherosclerotic plaque cells, but not by cells in normal arteries [ 34 - 37 ]. In addition, extensive inflammation and destruction of musculo-elastic vessel wall elements have been observed in dilated human coronary arteries [ 38 , 39 ]. Schoenhagen et al. [ 40 ] suggest that the degradation of extracellular matrix by MMP-3 may contribute to the expansion of the coronary vessel wall. This effect is characteristic of positive remodeling. Based on these and our own observations, we maintain that MMP-3 over-expression may occur in aneurysm segments. Histopathologic studies would be needed to clarify whether or not this is the case.MMP levels are elevated in patients with acute myocardial infarction, unstable angina and coronary angioplasty [ 35 , 41 , 42 ]. All patients in our study had been diagnosed with stable angina before being enrolled into the study.CRP reflects systemic inflammatory activity. In this study, we did not observe increased CRP levels in those patients with coronary aneurysms. One explanation for similar CRP expression between cases and controls might be that all study subjects had been diagnosed with stable angina pectoris.Varying degrees of inflammation are reported among individuals with abdominal aortic aneurysms. This variation may relate to possible confounding due to clinical manifestations (asymptomatic or symptomatic) and aneurysm progression rates (cm/year). Other investigators have failed to observe increased CRP levels among asymptomatic patients with abdominal aortic aneurysm [ 43 ].Because elevated MMP-3 levels likely contribute to the development of coronary aneurysms, this matrix-degrading enzyme may represent an important therapeutic target. Luan et al. [ 44 ] reported that a number of statins inhibit MMP-3 activity in rabbits. COX-2 inhibitors may also suppress MMP expression. Production of MMPs by macrophages occurs through a PGE 2 /cAMP-dependent pathway [ 45 ]. Theoretically, COX-2 inhibitors could attenuate this pathway. Another target of MMP inhibition has been demonstrated in animal models of adenovirus-mediated TIMP gene transfer [ 46 ].In reporting our findings, we acknowledge that measurement of TIMP levels between cases and controls would have provided useful information about the possibility of proteolytic imbalance. Similarly, measurement of locally produced inflammatory cytokines, hormones and growth factors would be interesting to know about, since these regulate matrix-degrading enzyme expression [ 16 - 19 ]. This could provide relevant information, as systemic inflammatory activity may not reflect local inflammatory infiltration in aneurysm segments. Finally, the study would have benefited from having a larger sample size as well as genotype determination.We conclude that MMP-3 overexpression due to a proteolytic imbalance may lead to coronary aneurysm development through degradation of matrix components, especially lamina elastica. New medical therapeutic options targeted specifically against MMP-3 may prove useful in the prevention of aneurysm formation.