Latent antithrombin does not affect physiological angiogenesis: An in vivo study on vascularization of grafted ovarian follicles

Latent antithrombin does not affect physiological angiogenesis: An in vivo study on vascularization of grafted ovarian follicles
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  Latent antithrombin does not affect physiological angiogenesis:An in vivo study on vascularization of grafted ovarian follicles Matthias W. Laschke a  , Zeynep Cengiz a  , Johannes N. Hoffmann  b ,Michael D. Menger  a  , Brigitte Vollmar  c, * a   Institute for Clinical & Experimental Surgery, University of Saarland, 66421 Homburg/Saar, Germany  b  Department of Surgery, Klinikum Grosshadern, Ludwig-Maximilians University, 81377 Munich, Germany c  Department of Experimental Surgery, University of Rostock, Schillingallee 70, 18055 Rostock, Germany Received 3 September 2003; accepted 3 December 2003 Abstract Latent antithrombin (L-AT), a heat-denatured form of native antithrombin (AT), is a potent inhibitor of  pathological tumor angiogenesis. In the present study, we have investigated whether L-AT has comparableantiangiogenic effects on physiological angiogenesis of ovarian tissue. For this purpose, preovulatory follicles of Syrian golden hamsters were mechanically isolated and transplanted into dorsal skinfold chambers chronicallyimplanted in L-AT- or AT-treated hamsters. Non-treated animals served as controls. Over 14 days after transplantation neovascularization of the follicular grafts was assessed in vivo by quantitative analysis of thenewly developed microvascular network, its microvessel density, the diameter of the microvessels, their red blood cell velocity and volumetric blood flow as well as leukocyte-endothelial cell interaction usingfluorescence microscopic techniques. In each group, all of the grafted follicles were able to induce angiogenesis.At day 3 after transplantation, sinusoidal sacculations and capillary sprouts could be observed, finallydeveloping complete glomerulum-like microvascular networks within 5 to 7 days. Overall revascularization of grafted follicles did not differ between the groups studied. Interestingly, follicular grafts in L-AT- and AT-treatedhamsters presented with higher values of microvessel diameters and volumetric blood flow, when compared tonon-treated controls, which may be best interpreted as a reactive response to an increased release of vasoactivemediators. In conclusion, the present study demonstrates, that L-AT has no adverse effects on physiologicalangiogenesis of freely transplanted ovarian follicles. Thus, L-AT may be an effective drug in tumor therapy, 0024-3205/$ - see front matter   D  2004 Elsevier Inc. All rights reserved.doi:10.1016/j.lfs.2003.12.008* Corresponding author. Tel.: +49-381-494-6220; fax: +49-381-494-6222.  E-mail address: (B. Vollmar). Life Sciences 75 (2004) 203–213  which blocks tumor growth by selective suppression of tumor vascularization without affecting new vesselformation in the female reproductive system. D  2004 Elsevier Inc. All rights reserved.  Keywords:  Latent antithrombin; Angiogenesis inhibitor; Physiological angiogenesis; Transplantation; Ovarian follicles;Intravital fluorescence microscopy; Tumor therapy Introduction Angiogenesis, the development and proliferation of new capillaries from pre-existing vessels, isa major prerequisite for the transition of tumors from a dormant state to a malignant state, because new vessels bring in oxygen, essential nutrients and growth factors allowing the tumor mass to expand (Folkman, 1989, 1990). This led to the idea that angiogenesis inhibitors may be used in cancer treatment  (Folkman, 1996). A number of such angiogenesis inhibitors have recently  been identified. Many of them are derived by modification of endogenous proteins which therebygain new properties, e.g. angiostatin, an analogous fragment of plasminogen (O’Reilly et al.,1994), or endostatin, a fragment produced by proteolytic cleavage of collagen XVIII (O’Reilly et  al., 1997).In 1997 Wardell described the preparative induction and structure of a novel modified endogenous protein called latent antithrombin (L-AT), a partially denaturated form of native antithrombin (AT)(Wardell et al., 1997). AT is a heparin-binding protein of the serpin superfamiliy and the principal  physiological inhibitor of thrombin and other serine proteases of the clotting cascade (Bone, 1992). Like all serpins, it traps its target proteinases by exposing a reactive loop, which is located on the surface of the enzyme (Carrell, 1999). Heat treatment transforms AT to the inactive L-ATwith low heparin affinity, in which the reactive loop is irreversibly inserted into the protein. Due to these conformational changescertain epitopes of L-AT can interact with appropriate endothelial components. Recently, L-AT has beenshown in vitro and in vivo to efficiently inhibit malignant angiogenesis and to suppress tumor growth(O’Reilly et al., 1999; Larsson et al., 2000). Thus, L-AT might be a promising antiangiogenic drug infuture tumor therapy.However, because angiogenesis is also involved in a number of physiological processeswithin the female reproductive system, e.g. folliculogenesis (Koos and LeMaire, 1983), corpus luteum formation (Gospodarowicz and Thrakal, 1978) and proliferation of the endometrium(Christianes et al., 1982), antiangiogenic tumor therapy may be associated with the risk toinduce side effects. These may include uterine and ovarian dysfunction, resulting in menstrualirregularities and iatrogenic infertility. Therefore it is necessary to further investigate the effectsof potential angiogenesis inhibitors such as L-AT not only specifically on new vessel formationin tumor growth, but also on physiological vascular proliferation within the female reproductivesystem.For this purpose, we analyzed in vivo the process of neovascularization and the microcirculationof ovarian follicles which were isolated from PMSG-stimulated Syrian golden hamsters and trans- planted into dorsal skinfold chambers of L-AT- or AT-treated animals using intravital fluorescencemicroscopy.  M.W. Laschke et al. / Life Sciences 75 (2004) 203–213 204  Methods  Preparation of the hamster dorsal skinfold chamber  The experiments were conducted in accordance with the German legislation on protection of animalsand the  NIH Guide for the Care and Use of Laboratory Animals  (Institute of Laboratory AnimalResources, National Research Council, Washington, USA).The chamber preparation contains one layer of striated muscle and skin and allows for intravitalmicroscopic observation of the microcirculation in the awake animals over a prolonged period of time.The chamber technique and its implantation procedure have been described previously in detail (Endrichet al., 1980). Briefly, under sodium pentobarbital anesthesia (50 mg/kg body weight i.p.), twosymmetrical titanium frames were implanted on the extended dorsal skinfold of 8- to 10-week oldSyrian golden hamsters (body weight, 60 to 80 g). One layer of skin was completely removed in acircular area 15 mm in diameter, and the remaining layers (consisting of striated skin muscle andsubcutaneous tissue) were covered with a removable cover slip incorporated into one of the titaniumframes. In addition, a permanent catheter was passed from the dorsal to the ventral side of the neck andinserted into the jugular vein for application of fluorescent dyes. The animals were allowed to recover from anesthesia and surgery for at least 48 h. After intravenous application of 0.2 ml of 5% fluoresceinisothiocyanate (FITC)-labeled dextran 150000 (Sigma, Deisenhofen, Germany), the chamber enabled for continuous observation and repetitive analysis of the microcirculation by means of intravital fluores-cence microscopy in the awake animals.  Follicle isolation and transplantation For follicle donation, four 8- to 10-week old female hamsters were used and pretreated with PMSG(Sigma) dissolved in PBS (1000 U/ml) in order to increase the number of preovulatory follicles. PMSGwas given subcutaneously at 8:00 a.m. at a single dose of 2 U/10 g body weight. After 48 h, donor ovaries were aseptically removed and placed in 30 mm diameter plastic Petri dishes, filled with 37 j CDMEM medium (10% fetal calf serum, 0.1 mg/ml gentamycin) and the fluorescent dye bisbenzimideH33342 (200  A g/ml) (Sigma). After removing the surrounding tissue, the ovaries were microdissectedunder a stereomicroscope using 27 gauge needles. According to size, the follicles were visually collectedand transferred into 37 j C bisbenzimide H33342-free DMEM medium, as described previously in detail(Vollmar et al., 2001).Follicles were transplanted into 8- to 10-week old, non-ovariectomized recipients. All recipients weresynchronized, i.e. in the same stage of the estrous cycle, in that transplantation was performed at day twoafter PMSG treatment, which represents the switch of di-estrous to pro-estrous stage of the 4-day cyclein the hamster. For follicle transplantation the cover glass of the skinfold chamber on recipient animalswas removed and one or two follicles were placed onto the striated muscle within each chamber. A hand- picking procedure guaranteed single follicles free of connective tissue for transplantation.  Intravital microscopy For in vivo microscopic observation, the awake animals were immobilized in a Plexiglas tube and thedorsal skinfold preparation was attached to the microscopic stage. Intravenous injection of 0.2 ml of 5%  M.W. Laschke et al. / Life Sciences 75 (2004) 203–213  205  FITC-labeled dextran 150000 (Sigma) guaranteed contrast enhancement by staining of plasma.Rhodamine 6G (0.1%, 0.1 ml i.v.; Sigma) allowed for the direct in vivo staining of leukocytes.Intravital microscopy was performed using a modified Leitz Orthoplan microscope with a 100W HBOmercury lamp attached to a Ploemo-Pak illuminator with blue, green and ultraviolet filter blocks (Leitz,Wetzlar, Germany) for epi-illumination. The microscopic images were recorded by a charge-coupleddevice video camera (CF8/1 FMC; Kappa GmbH, Gleichen, Germany) and transferred to a video systemfor off-line evaluation. With the use of    4,   6.3,   10 and   20 long distance objectives (Leitz),magnifications of    86,   136,   216 and   432 were achieved on a 14-inch video screen (PVM 1444;Sony, Tokyo, Japan).  Microcirculatory analysis By means of the computer-assisted microcirculation analysis system CapImage (Zeintl, Heidelberg,Germany) quantitative off-line analysis of the videotapes included the determination of the diameter ( A m) and the size of the transplanted follicles (mm 2 ), the size of the growing microvascular networks (in% of the initial follicular size), the microvessel density, i.e. the length of red blood cell (RBC)-perfusedmicrovessels per observation area (cm/cm 2 ) and the diameters of the newly developing follicular microvessels ( A m).The diameter and the size of the transplanted follicles were determined with the use of the bisbenzimide fluorescence. On ultraviolet epi-illumination the dye bisbenzimide is characterized by a bright blue fluorescence with only little bleaching that persists through several cell generations. Thespecific fluorescence/background fluorescence ratio is high enough throughout a period of 2 weeks to precisely delineate the stained follicular graft from the surrounding unaffected host tissue. The area of fully developed microvascular networks might sometimes slightly exceed the follicular tissue area withthe consequence that values of the size of the growing microvascular networks are >100% of thefollicular size (Laschke et al., 2002, 2003).The microvessel density was assessed by the length of red blood cell (RBC)-perfused microvessels per observation area. In contrast to morphological studies from cross-sectioned tissue, which evaluatevessel density by determining the number of vessels per observation area, the measure of perfusedmicrovessels per observation area is a highly accepted parameter in in vivo microcirculation research(Schmid-Schoenbein et al., 1977; Nolte et al., 1995). In the present study, we preferred to use the in vivoapproach, because it allows repeated analysis of the identical tissue over time. In addition, the resultsmay have higher relevance, because they distinguish between perfused and non-perfused microvessels,estimating the true fraction of newly formed vessels that contribute to nutritional supply (Vajkoczy et al.,1998, 1999).Centerline RBC velocity (V RBC ) in the individual capillaries was measured by frame-to-frameanalysis. Volumetric blood flow (VQ) of individual capillaries was calculated from V RBC  and diameter (D) for each microvessel as VQ =  k    (D/2) 2   V RBC /K, where K (=1.3) represents the Baker/ Wayland factor  (Baker and Wayland, 1974), considering the parabolic velocity profile of blood in microvessels.Rhodamine 6G-stained leukocytes were classified in accordance to their interaction with theendothelium of newly formed microvessels. Rolling cells were defined as cells moving with a velocityless than two-fifths of the centerline velocity (given as percentage of nonadherent leukocytes passingthrough the observed vessel segment within 20 seconds). Adherent cells were defined as cells that did  M.W. Laschke et al. / Life Sciences 75 (2004) 203–213 206  not move or detach from the endothelial lining during an observation period of 20 seconds (given asnumber of cells per microvascular network area).  Experimental protocol  A first group of 5 female hamsters were treated with L-AT (25 mg/kg body weight/d, s.c.; AventisBehring, Marburg, Germany), whereas a second group (n = 6) received AT (25 mg/kg body weight/d,s.c.; Aventis Behring). This dosage of L-AT has been shown to almost completely block tumor growth(O’Reilly et al., 1999). Treatment with ATand L-ATstarted at the day of follicle transplantation, and wasgiven daily throughout the entire 14-days observation period. The drugs were injected subcutaneouslyinto the left flank of the animals. Non-treated animals (n = 4) served as control. In each experimentalgroup angiogenesis of 8 follicles was analyzed. All animals were synchronized with PMSG.The macroscopic appearance of the skinfold chamber preparation and the implanted grafts weredocumented daily. Intravital microscopic analysis of the follicular grafts was performed on days 3, 5, 7,10 and 14 after transplantation. Microvessel density was measured within five regions of interest per graft and observation time point. Regions of interest were selected along a virtual horizontal overlayedon the individual follicle. Microvascular diameters and hemodynamic parameters were determined byanalyzing ten microvessels per region of interest. Microvessels were selected randomly inasmuch asthose microvessels were chosen for analysis of diameter which crossed a vertical line drawn over thecenter of the video screen. In all microvessels selected, both vessel diameter and V RBC  were determinedfor subsequent calculation of VQ. Statistics After multivariate analysis of interaction between time and group, differences between groups weretested separately at each time point by ANOVA followed by the appropriate post hoc comparison.Differences within the groups over time were analyzed by ANOVA for repeated measurements and werefollowed by appropriate multiple post-hoc comparisons. A P value < 0.05 was considered significant.All values are expressed as means  F  S.E. Results All of the isolated follicles were able to induce angiogenesis after transplantation into the dorsalskinfold chamber, regardless of whether they had been grafted in L-AT-treated, AT-treated or non-treatedanimals. In each experimental group, the development of new microvascular networks within thefollicular grafts followed the same characteristic stages. Typical signs of angiogenesis could be observedat day 3. These included sinusoidal sacculations and capillary sprouts, srcinating from the host striatedmuscle capillaries and postcapillary venules. At this time, follicles were already vascularized toapproximately 50 to 70% (Figs. 1 and 2). During the following days, these sprouts interconnected with each other and finally developed complete microvascular networks, exhibiting follicular network sizesof   f 120 to  f 130% at day 14 (Fig. 2). Due to their glomerulum-like appearance they could be clearly distinguished from the striated muscle microvessels of the dorsal skinfold chamber, which arecharacterized by a typical parallel arrangement  (Menger and Lehr, 1993).  M.W. Laschke et al. / Life Sciences 75 (2004) 203–213  207
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