Elastin Stabilization for Treatment of Abdominal Aortic Aneurysms Elastin Stabilization for Treatment of Abdominal Aortic Aneurysms Jason C. Isenburg, PhD*; Dan T. Simionescu, PhD*; Barry C. Starcher, PhD; Narendra R. Vyavahare, PhD Background—Maintaining the integrity of arterial elastin is vital for the prevention of abdominal aortic aneurysm (AAA) development. We hypothesized that in vivo stabilization of aortic elastin with pentagalloyl glucose (PGG), an elastin-binding polyphenol, would interfere with AAA development. Methods and Results—Safety and efficacy of PGG treatment were first tested in vitro using cytotoxicity, elastin stability, and PGG-elastin interaction assays. For in vivo studies, the efficacy of PGG was evaluated within a well-established AAA model in rats on the basis of CaCl2-mediated aortic injury. With this model, PGG was delivered periadventitially at 2 separate time points during the course of AAA development; aortic diameter, elastin integrity, and other pathological aspects were monitored and evaluated in PGG-treated aortas compared with saline-treated control aortas. Our results show that a one-time periadventitial delivery of noncytotoxic levels of PGG inhibits elastin degeneration, attenuates aneurysmal expansion, and hinders AAA development in rats without interfering with the pathogenic mechanisms typical of this model, namely inflammation, calcification, and high metalloproteinase activities. PGG binds specifically to arterial elastin and, in doing so, preserves the integrity of elastic lamellae despite the presence of high levels of proteinases derived from inflammatory cells. Conclusions—Periadventitial administration of PGG hinders the development of AAA in a clinically relevant animal model. Stabilization of aortic elastin in aneurysm-prone arterial segments offers great potential toward the development of safe and effective therapies for AAAs. (Circulation. 2007;115:1729-1737.) Key Words: aneurysm � aorta � drug delivery systems � elasticity � metalloproteinases � prevention � tannins Abdominal aortic aneurysms (AAAs) are associatedwith impaired arterial wall integrity, leading to ab- normal ballooning and eventual fatal rupture. AAAs, which are apparently increasing in frequency, have been cited as one of the top 10 causes of death among older men.1 After initial diagnosis, AAA patients (AAA diame- ter ��2.5 cm) are monitored for an increase in aortic diameter, and surgery aimed at preventing death from enlarged or ruptured AAAs is recommended when the diameter reaches ��5.5 cm.2 No pharmacological treat- ment currently exists for AAAs. Surgical procedures entail either endovascular stent graft repair or complete replace- ment of the diseased aortic segment with an artificial vascular graft. Although often effective, endovascular stents are anatomically appropriate for only 30% to 60% of AAA patients at the outset and present the risk of en- doleaks and graft displacement.3 Moreover, open surgery for full-size graft insertion is highly invasive, limiting its use to those patients with high operative risk. Clinical Perspective p 1737 Treatment options are particularly limited for patients with small or moderate aneurysms; this group makes up the largest percentage of all AAA patients.4 This number is likely to increase dramatically with the advent of “blanket” screening of asymptomatic subjects with imaging surveillance.5 Conse- quently, novel therapeutic approaches targeted at hindering the progression of AAAs promptly after diagnosis would be extremely beneficial. Information about AAA mechanisms is currently gathered from analysis of late-stage aneurysmal samples obtained from patients and animal models. In addition to arterial dilatation, AAAs are characterized by degeneration of the arterial architecture,6 decreased medial elastin content,7 disruption or fragmentation of elastic lamellae,8 presence of matrix- degrading enzymes such as matrix metalloproteinases (MMPs),9,10 inflammatory infiltration,10 and often calcifica- tion.11 As a result of its multifactorial pathogenesis, antiin- Received November 3, 2006; accepted January 26, 2007. From the Department of Bioengineering (J.C.I., D.T.S., N.R.V.) Clemson University, Clemson, SC, and Department of Biochemistry, University of Texas Health Center, Tyler (B.C.S.). The online-only Data Supplement, consisting of expanded Methods, is available with this article at http://circ.ahajournals.org/cgi/ content/full/CIRCULATIONAHA.106.672873/DC1. *Dr Isenburg and Dr Simionescu contributed equally to this work. Correspondence to Narendra R. Vyavahare, PhD, Cardiovascular Implant Research Laboratory, Department of Bioengineering, Clemson University, 501 Rhodes Engineering Research Center, Clemson, SC 29634. E-mail narenv@clemson.edu © 2007 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.106.672873 1729 D ow nloaded from http://ahajournals.org by on A pril 5, 2021 flammatory agents,12 proteinase inhibitors (such as tissue inhib- itors of metalloproteinases [TIMPs]), and genetic and pharmacological inhibition of MMPs13 have been tested as potential AAA treatments in experimental animals, but none has yet reached clinical application. Because long-term, adequate control of local inflammation and MMP activities may be difficult to achieve and may be accompanied by adverse side effects,14 our hypothesis was that stabilization of aortic elastin in aneurysm-prone arterial segments offers potential for the devel- opment of safe and effective therapies for AAAs. In current studies, we have explored polyphenolic tannins, specifically pentagalloyl glucose (PGG), as novel elastin- stabilizing agents. Tannins bind to elastin15 and, in doing so, render it resistant to enzymatic degradation.15–17 We provide evidence that periarterial treatment with PGG preserves elastin fiber integrity and hinders aneurysmal dilatation of the abdom- inal aorta in a clinically relevant animal model of AAA. Methods Please see the online Data Supplement for expanded Methods. Pentagalloyl Glucose High-purity PGG used throughout the in vitro and in vivo experi- ments was produced from tannic acid by methanolysis, as described previously.17 In Vitro Cytotoxicity Primary rat aortic smooth muscle cells and rat skin fibroblasts were exposed to increasing concentrations of PGG and viability assessed by the soluble tetrazolium salt (MTS) assay (Promega, Madison, Wis) and Live-Dead staining (Molecular Probes, Eugene, Ore). Cells also were incubated with PBS and 70% ethanol as negative and positive controls, respectively. In Vitro Efficacy Native abdominal aortas collected from adult rats were treated in vitro with increasing concentrations of PGG dissolved in saline. Control samples were treated with saline alone. Aortic samples were tested for resistance to elastase by desmosine analysis.15,18 To demonstrate PGG– aorta interactions, samples were stained with a phenol-specific histology stain and were tested for natural recoil ability using opening-angle measurements, as described previously.17,19 Animal Surgeries Two experiments were designed to evaluate the in vivo efficacy of PGG in hindering AAA formation and progression in a CaCl2 injury model of aortic aneurysm (Figure 1). Experiment 1: Interference With Formation of Early AAA Infrarenal abdominal aortas of adult male Sprague-Dawley rats were perivascularly treated for 15 minutes with PGG dissolved in physi- ological saline using a presoaked gauze applicator. After rinsing, the aortas underwent chemical injury by application of 0.5 mol/L CaCl2 for 15 minutes. Control aortas were treated with vehicle (saline) for 15 minutes, rinsed, and then subjected to CaCl2. After 28 days, rats were humanely euthanized with CO2 asphyxiation, and aortas were excised and analyzed as described below. Changes in external aortic diameter were measured by digital photography. Experiment 2: Hindrance of AAA Progression Abdominal aortas were treated perivascularly 0.5 mol/L CaCl2 to induce AAA, and rats were allowed to recover. After 28 days, the aneurysmal aortas were reexposed and treated with PGG in saline for 15 minutes, as described above. In the control group, aneurysmal aortas were treated with vehicle (saline) for 15 minutes. At 28 days after the second surgery (56 days after initial CaCl2 injury), rats were humanely euthanized, and samples were collected for analysis. Changes in external aortic diameter were measured by digital photography. All animals used in our experiments received humane care in compliance with protocols approved by the Clemson University Animal Research Committee as formulated by the National Institutes of Health (publication No. 86-23, revised 1999). Elastin Content and Integrity Assessment Aortic segments were analyzed for desmosine content by radioim- munoassay18 and by histology using Verhoeff van Gieson stain for elastin and hematoxylin and eosin for general structure. PGG Binding Studies To extract PGG, aortas retrieved 28 days after PGG treatment (experiment 1) were pulverized and shaken with 80% methanol; extracts were assayed for phenol content with the Folin-Denis reaction.20 As positive controls, rat aortas were treated with PGG in situ for 15 minutes and extracted for analysis of initial PGG content. Calcification Assessment Rat aortic samples retrieved at 28 days after CaCl2 injury (experi- ment 1) were analyzed for calcium content using atomic absorption spectrophotometry and by histological Alizarin red staining, as described before.21 MMP and TIMP Quantification MMP-2 and MMP-9 activities in rat aortas retrieved 28 days after CaCl2 injury (experiment 1) were analyzed by gelatin zymography.22 TIMP-2 levels were assayed by ELISA (Amersham Biosciences, Piscataway, NJ). Immunohistochemistry Macrophages were detected on samples from control (saline) and PGG-treated aortas 28 days after treatment (experiment 1) using a mouse anti-rat monocyte/macrophage primary antibody (Chemicon International, Temecula, Calif), whereas lymphocytes were detected with a rabbit anti-CD3 T-cell primary antibody (Sigma-Aldrich, St Louis, Mo). Staining was performed with the appropriate biotinyl- ated secondary antibodies and an avidin-biotin detection system (Vector Laboratories, Burlingame, Calif). Figure 1. Experimental design for in vivo experiments. In experi- ment 1, PGG was applied to healthy abdominal aorta immedi- ately before CaCl2-mediated injury, and AAA development was followed for 28 days. In experiment 2, PGG was applied to an- eurysmal aorta, and development of advanced AAA was moni- tored for another 28 days. 1730 Circulation April 3, 2007 D ow nloaded from http://ahajournals.org by on A pril 5, 2021 Statistical Analyses Results are expressed as mean�SEM. Statistical analyses of the data were performed using single-factor ANOVA. Subsequently, differ- ences between means were determined through the use of the least significant difference with an � value of 0.05. The authors had full access to and take responsibility for the integrity of the data. All authors have read and agree to the manuscript as written. Results In Vitro Safety and Efficacy Studies In preparation for the animal studies, several experiments were performed in vitro to evaluate the effects of a single 15-minute application of PGG on cells and arterial extracel- lular matrix, specifically elastin. MTS results (Figure 2A and 2B) showed that exposure of cells to PGG concentrations of up to 0.06% had minimal cytotoxic effects. No statistical differences were observed between cells exposed to 0.03% and 0.06% PGG (P�0.05). These results were also confirmed by Live-Dead assay (data not shown). Desmosine analysis of aortic samples exposed to elastase showed an increasing trend in elastin preservation with increasing PGG concentrations (Figure 2C). Moreover, the intensity of polyphenol histolog- ical staining increased progressively with increasing concen- trations of PGG, revealing direct binding of PGG to elastic fibers (Figure 2D). For further proof of PGG-arterial elastic fiber interactions, the ring-opening test was performed on PGG-treated native rat aortic rings (Figure 2E and 2F). When allowed to open by a single incision, rings extended to �75°, revealing the natural elastic recoil properties of aortic tissues. Rings that were exposed to increasing concentrations of PGG exhibited progressively smaller opening angles, suggestive of the direct interactions of PGG with elastic fibers. Having established that a 15-minute exposure to PGG solutions of up to 0.06% is not cytotoxic and effectively stabilizes aortic tissue by binding to elastic fibers, we de- signed 2 experiments to test the in vivo efficacy of PGG in hindering AAA formation and progression (Figure 1). Perivascular Delivery of Noncytotoxic Levels of PGG Hinders Aneurysm Formation In experiment 1, perivascular application of CaCl2 to the infrarenal abdominal aorta induced significant changes in aortic diameter at 28 days after injury (Figure 3A and 3B). Figure 2. In vitro safety and efficacy studies of PGG interactions with native rat abdominal aorta. Rat aortic smooth muscle cells (A) and rat skin fibroblasts (B) were exposed to increasing concentrations of PGG, and cytotoxicity was measured with the MTS assay (n�3 per group per cell type). Cells exposed to PBS and 70% ethanol (EtOH) were used as negative and positive controls, respectively. C, To test resistance to elastase, aortic samples were exposed to increasing concentrations of PGG for 15 minutes in vitro and then exposed to pure elastase. Aortic elastin content was measured in each group by desmosine analysis (n�5 per group). D, For histological confirmation of PGG binding to aortic elastin, PGG-treated aortic tissues were stained with a phenol-specific stain (black indicates PGG) and counterstained with light green (L indicates lumen; bar�50 �m). Arrows point to elastic lamellae. E, To test elastic recoil, aortic rings were treated for 15 minutes with increasing concentrations of PGG and subjected to ring-opening analysis (n�5 per group). Opening angles were measured graphically (bot- tom left corner). F, Mean opening angles for each group are shown as a function of PGG concentration. *Statistically significant differences (P�0.05) relative to the negative control group. Isenburg et al Aneurysm Treatment by Elastin Stabilization 1731 D ow nloaded from http://ahajournals.org by on A pril 5, 2021 Comparative measurements of control rats at days 0 and 28 after surgery (1.3�0.05 and 1.9�0.1 mm, respectively) revealed a mean increase in diameter of 42�10% (P�0.05; n�12). In comparison, aortas exposed to PGG exhibited a minimal (8�7%) increase in diameter after 28 days (from 1.5�0.06 to 1.6�0.09 mm). With an arbitrary threshold of a 20% diameter increase considered aneu- rysmal in this experimental model, 8 of 12 rats exhibited aneurysms in the control group (66.7%), and only 2 of 11 rats were aneurysmal in the PGG group (18.2%; Figure 3C). These results indicate that PGG application to healthy aorta effectively hindered formation of aneurysms in this experimental model. As shown above, exposure to PGG had minimal in vitro cytotoxic effects on rat cells. In our animal studies, PGG- treated rats did not exhibit significant weight losses during the 28-day test period (42.3�3.4 g gain in the control group versus 44.2�3.6 g in the PGG group; P�0.05; n�12). Liver samples did not exhibit any noticeable histological changes indicative of hepatotoxicity (data not shown). In addition, levels of serum ALT (an enzyme used to assess liver function) for saline controls (9.1�1.5 U/L) and PGG-treated rats (17.7�3.1 U/L) were consistently within the acceptable range (5 to 45 U/L). Furthermore, ALT levels for PGG- treated rats were not statistically different from those of nonsurgery controls rats (11.5�1.2 U/L; P�0.05), suggesting Figure 3. Local delivery of PGG prevents AAA development and elastin degeneration. Adult rat abdominal aortas were treated periad- ventitially with PGG for 15 minutes, rinsed, and then exposed to CaCl2-mediated chemical injury to induce aneurysm formation. In con- trol rats, PGG treatment was replaced with saline, followed by CaCl2. A, External diameter of the infrarenal aorta between the renal artery (top) and iliac bifurcation (bottom) was measured by digital photography. Black dashed lines were added to aid in the identifica- tion of aortic anatomy. B, Mean percent change in diameter at 28 days relative to day 0 (n�12 for saline controls, n�11 for PGG). C, Calculated aneurysm occurrence using an arbitrary 20% diameter increase threshold for defining AAA. D, Elastin integrity evaluated by histology using VVG stain (black L indicates lumen; elastin stained black; bar � 50 �m) and (E) desmosine analysis. F, Quantitative PGG content analysis in explanted aorta (n�3). Thick black arrows in B and E designate timing of PGG application. *Statistically signifi- cant differences between means (P�0.05) at 28 days relative to saline-treated controls. 1732 Circulation April 3, 2007 D ow nloaded from http://ahajournals.org by on A pril 5, 2021 that PGG treatment did not induce major liver damage within this model. Treatment With PGG Prevents Degeneration of Aortic Elastin In parallel with aortic dilatation, experimentally induced AAAs were accompanied by major changes in vascular elastin content and integrity as shown by desmosine analysis and histology (Figure 3D and 3E). Compared with nonsur- gery control aorta, aortic elastin content in the control group diminished by almost 50% (Figure 3E) and exhibited char- acteristic flattening and fragmentation of the elastic laminae (Figure 3D) at 28 days after injury. Conversely, aortas from the PGG group exhibited minimal (�15%) decrease in elastin content (Figure 3E) and excellent preservation of elastic laminar integrity and waviness (Figure 3D), suggesting that PGG delivery effectively prevented elastin degeneration in this animal model. PGG Binds to Aortic Elastin In Vivo The affinity of phenolic tannins toward elastin is well known from histology techniques23 and was previously demonstrated in vitro with pure elastin.15 Compared with day 0 (1.8�0.6 �g PGG/mg dry tissue), aortas explanted 28 days after PGG application contained slightly lower (Figure 3F) but not statistically different amounts of PGG (1.2�0.4 �g PGG/mg dry tissue; P�0.05), indicating that in vivo binding of PGG to aortic tissues is stable for a minimum of 28 days in this animal model. Treatment With PGG Does Not Interfere With Model-Related Pathogenesis To investigate pathogenic aspects typical to this animal model,24 calcium content, macrophage infiltration, MMP activities, and expression of TIMP were analyzed in aortas retrieved 28 days after CaCl2 treatment (experiment 1). Perivascular injury induced significant tissue calcification after 28 days (Figure 4A), which was localized mainly in the media (Figure 4B). Similar calcium values (P�0.05) and distribution were observed in PGG-treated aortas. MMP activities and TIMP-2 levels (Figure 4C) also were not different between controls and PGG-treated aortas (P�0.05), suggesting that tissues in both groups were exposed to similar proteolytic environments. Moreover, hematoxylin and eosin and immunohistochemical staining for macrophages and lymphocytes revealed comparable inflammatory infiltrates in both groups (Figure 4D). Taken together, these results sug- gest that PGG treatment did not interfere with key pathogenic mechanisms typical of this AAA experimental model. Periadventitial Treatment of Aneurysmal Aorta With PGG Limits AAA Progression In experiment 2, rat aortas were treated with CaCl2, and AAA was allowed to develop for 28 days. At this time point, the Figure 4. PGG application does not interfere with pathogenic mechanisms of AAA formation. A, Aortic calcification at 28 days after CaCl2 injury was evaluated by calcium content analysis (n�12 per group) and (B) histology using Alizarin red stain (red L indicates lumen; calcium deposits stained red). C, MMP-2 and MMP-9 enzymatic activities and TIMP-2 levels in the 2 groups were not statisti- cally different (P�0.05; n�6 per group) at 28 days after injury. D, Immunohistochemistry for macrophages (bar�100 �m) and lympho- cytes (bar�50 �m). For both types, positive reaction is brown. Isenburg et al Aneurysm Treatment by Elastin Stabilization 1733 D ow nloaded from http://ahajournals.org by on A pril 5, 2021 aneurysmal aortas were exposed through a second surgery, and PGG was applied perivascularly (PGG group). Saline was applied to aneurysmal aortas in the control group. AAA progression was followed for another 28 days in both groups. A progressive diameter increase, reaching a mean 47.1�11% increase at 56 days, was measured in the control group (Figure 5A and 5B). Approximately half of the aneurysmal aortas significantly increased in diameter from day 28 to 56, indicating chronic AAA progression in this animal model (Figure 5C). Conversely, aneurysmal aortas that were exposed to PGG exhibited no increase in mean diameter at 56 days compared with day 28 mean values (Figure 5B). It is especially noteworthy that 100% of aortas in the PGG group (11 of 11) maintained the same diameter or exhibited a decrease in aortic diameter at 56 compared with 28 days (Figure 5C). The mean diameter value at 56 days for the PGG group was actually slightly lower than that at 28 days but not statistically significant (P�0.05). Overall, these results indicate that PGG application to aneurysmal aortas effectively hindered arterial dilatation in this experimental model. Effect of PGG on Degeneration of Aortic Elastin At 56 days after injury, aneurysmal aortas exhibited extensive flattening, fragmentation, and degeneration of the elastic laminae in the control group (Figure 5D). Overall tissue architecture was indicative of severe tissue degeneration as outlined by numerous gaps or lacunae, bestowing the aneu- rysmal aorta with a porous, “spongy” aspect. In contrast, PGG-treated aortas exhibited improved preservation of elas- tic laminar integrity and waviness and overall preserved tissue architecture (Figure 5D). Analysis of vascular elastin revealed no significant differences in elastin content as shown by desmosine analysis (679.5�63.2 and 715.9�90.6 pmol desmosine/mg dry tissue). Moreover, these elastin values were not statistically different from the 28-day values Figure 5. Delivery of PGG to aneurysmal aorta prevents AAA progression. Adult rat abdominal aortas were treated periadventitially with CaCl2, and AAAs were allowed to develop for 28 days. At that time, aneurysmal aortas were reexposed and treated with PGG. In con- trol rats, PGG treatment was replaced with saline. Rats in both groups were euthanized 28 days after the second surgery (total, 56 days after initial CaCl2 injury). A, External diameter of the infrarenal aorta was measured by digital photography after perfusion fixation. Black dashed lines were added to aid in the identification of aortic anatomy. B, Mean percent change in diameter at 28 and 56 days relative to day 0 (n�11 per group). Thick black arrow designates timing of PGG application. C, Numbers of individual rats that exhib- ited increased, stationary, or reduced aortic diameters at 56 vs 28 days. D, Elastin integrity and tissue architecture were evaluated by histology using VVG stain (black L indicates lumen; elastin stained black) and hematoxylin and eosin. Bar�40 �m. E, Low-magnification pictures used to digitally measure the thickness of arterial media. Bar�1000 �m. *Statistically significant differences between means (P�0.05). 1734 Circulation April 3, 2007 D ow nloaded from http://ahajournals.org by on A pril 5, 2021 (503.0�69.2 pmol desmosine/mg dry tissue; P�0.05), indi- cating that the severe elastin degradation observed at 28 days has reached a steady state and has not changed significantly as AAA progressed from 28 to 56 days. Morphometric measurements on perfusion-fixed aortas (Figure 5E) revealed that the thickness of the aortic media was significantly smaller in the PGG treatment group (55.4�2.4 �m) com- pared with saline-treated aneurysmal controls (80.3�4.7 �m; P�0.05). Taken together, these results suggest that PGG treatment of aneurysmal aortas effectively prevented chronic diameter expansion and aortic matrix deterioration in this AAA animal model. Discussion Aneurysms arise from irreversible, chronic pathological changes that involve degeneration of structural matrix com- ponents. The role of MMP/TIMP imbalances in AAA initia- tion and development has been irrevocably demonstrated by studies using MMP- and TIMP-knockout mice.25–27 MMP- mediated loss of elastin, a hallmark of AAA, is believed to lead to progressive dilatation of the aorta. Therefore, our studies focused on chemical stabilization of vascular elastin as a novel approach to limit aneurysmal degeneration. The traditional approach to treating AAAs involves surgi- cal procedures such as endovascular stent graft repairs or complete replacement of the diseased section of the aorta. Nonsurgical approaches, currently still in the experimental phase, include use of antiinflammatory agents, proteinase inhibitors, and genetic and pharmacological inhibition of MMPs in animal models. Doxycycline, an antibiotic and MMP inhibitor, showed initial promising results in a pilot clinical study.28 Recently, Yoshimura et al29 reported that the systemic pharmacological inhibition of c-Jun N-terminal kinase, an intracellular signaling switch that controls MMP production, might block AAA progression and stimulate aneurysm regression. Although numerous attempts have focused on proteolytic inhibition for AAA treatment, we present a radically innova- tive approach, namely periadventitial delivery of noncyto- toxic concentrations of phenolic tannins such as PGG to stabilize elastin and render it resistant to enzymatic degrada- tion. In previous studies, we have shown that tannic acid (a decagalloyl glucose) binds to pure aortic elastin and porcine aortic wall, in both cases resulting in improved resistance to enzyme-mediated elastin degradation.15,16 More recently, we have reported that PGG, a more stable and less cytotoxic derivative of tannic acid, also binds to and protects elastin within porcine aortic wall with very similar efficacy.17 Our hypothesis was that the ability of PGG to stabilize elastin would extend in vivo to rat aorta. To verify our hypothesis, we performed a series of in vitro safety and efficacy studies, followed by in vivo validation in a rat AAA model. Safety and Efficacy Studies Results showed that PGG binds specifically to vascular elastin and, in doing so, renders elastin resistant to enzymatic degradation. In addition to being an effective elastin- stabilizing agent, PGG, when used at concentrations of up to 0.06%, had virtually no adverse effects on cell viability when applied in vitro to rat cells. Local PGG Delivery Hinders AAA Formation and Limits Diameter Increase Traditionally, an increase in aortic diameter is considered the defining characteristic of AAA. Numerous investigators stud- ied AAA formation in the CaCl2 injury model in mice,30 rabbits,11,31 and rats.32 We have previously used this model to show that CaCl2 injury induces a series of pathogenic alter- ations that share many similarities to human pathology and to changes observed in other experimental animals.24 In exper- iment 1, application of CaCl2 to the rat infrarenal abdominal aorta induced a mean increase in diameter of 42% at 28 days after application. The extent of aneurysmal dilatation ob- served in our studies corresponds well with other published studies using CaCl2 injury that consistently reported a 25% to 75% increase in diameter in rodents.25,26,32,33 In our studies, this pathology was observed in 8 of 12 control rats (66% incidence). This prevalence also was similar to other studies that reported AAA incidences of 60% to 80%.25,26 It is unknown why a consistent number of genetically similar experimental animals are apparently resistant to CaCl2- mediated injury. This aspect warrants further investigation. Conversely, mean aortic diameter increase in the PGG group was �10%, which is traditionally not considered indicative of aneurysm formation. The overall AAA inci- dence in the PGG group was �20%, indicating that in this model, PGG prevented aneurysm formation in more than 8 of 10 animals. Although the detailed mechanisms of this suc- cessful approach are not entirely known, we provide evidence that it likely involves PGG-mediated elastin stabilization. In addition to dilatation, AAAs also are characterized by progressive damage to arterial elastin. In our experimental model, periadventitial application of CaCl2 induced a mean decrease of �50% in aortic elastin content. Earlier studies have shown that elastin degeneration starts within the first days after CaCl2 injury and that this chronic process is related to an overexpression of MMPs within the aortic extracellular matrix.24 Similar to diameter increase, the incidence of elastin degeneration in CaCl2-treated aorta was observed in �80% of control rats. Typically, aortic samples that were characterized by low elastin content also exhibited characteristic flattening and fragmentation of the naturally wavy elastic lamellae. This histological aspect is characteristic of the aortic pathology in CaCl2-treated aorta.29 It is not clear at this time what the mechanism and clinical significance of elastic fiber flattening are, and this aspect merits further examination. Periadventitial treatment of aorta with PGG resulted in remarkable preservation of elastin integrity. Quantitative desmosine results showed that elastin content in the PGG group was not statistically different from the nonsurgery controls (P�0.05), indicating minor, if any, elastin degrada- tion. This was accompanied by excellent histological preser- vation of elastic lamellar integrity and waviness. Elastin preservation in the CaCl2 experimental model also was observed in aortas of MMP-knockout mice25,26 and in mice treated systemically with a c-Jun N-terminal kinase inhibi- tor.29 In all studies published thus far, the present report Isenburg et al Aneurysm Treatment by Elastin Stabilization 1735 D ow nloaded from http://ahajournals.org by on A pril 5, 2021 included, elastin preservation was consistently associated with the absence of aneurysm initiation or progression. This indicates that elastin stabilization by either MMP inhibition or PGG treatment may be effective as a potential treatment of AAAs. Periadventitial Treatment of Aneurysmal Aorta With PGG Limits AAA Progression In a more clinically relevant experiment, PGG was directly applied to aneurysmal aortas, and AAA progression was fol- lowed after this surgical intervention. Although most control aneurysmal aortas (saline treated) continued to expand in diam- eter, all of the PGG-treated aneurysmal aortas maintained the same diameter or exhibited a decrease in aortic diameter at the end of the experiment (56 days). Diameter expansion in aneu- rysmal controls was associated with changes in medial thickness and integrity that included severe tissue degeneration. Yo- shimura et al29 reported similar inhibition of AAA progression after aneurysm onset in rodent animal models as a result of systemic pharmacological inhibition of c-Jun N-terminal kinase. Because c-Jun N-terminal kinase is an intracellular signaling switch that controls MMP production, its inhibition hindered MMP production and thus prevented degeneration of the vascu- lar matrix. In our studies, histological analysis of aneurysmal aortas at day 56 showed a significant decline in elastin integrity in the control group, indicating that elastin degeneration is a chronic pathogenic aspect characteristic of this AAA model. Hallmarks of this process were elastic fiber flattening, fragmentation, and lack of elastic laminar staining in extended areas in almost all samples (5 of 6). Aortas from the PGG group exhibited im- proved preservation of elastic laminar integrity and waviness in most samples (4 of 6). Desmosine analysis for this experiment revealed little quantitative difference for the PGG group com- pared with controls, possibly because elastin degradation has reached a maximum threshold at 28 days. Being mindful of these limitations, we nonetheless suggest that PGG-mediated elastin stabilization in aneurysmal aortas has the potential to significantly hinder AAA development. Mechanisms of PGG-Mediated Elastin Stabilization As demonstrated before, PGG exhibits an outstanding affinity toward elastin, possibly binding to hydrophobic areas, which are known to be susceptible to protease-mediated elastolysis.16 In the present studies, we have validated PGG binding to aortic elastin in vivo and provided evidence that the binding is stable for a minimum of 28 days in this accelerated AAA experimental model. We also have provided ample in vitro evidence that binding of PGG to arterial elastin provides an outstanding resistance to proteolytic degeneration.17 Because natural elastin turnover is exceptionally low,34 we hypothesize that PGG may remain bound to vascular elastin for extended periods of time after application, sufficient to maintain resistance to enzymes and to deter AAA progression. However, more studies are required to validate this hypothesis. The mechanisms of aneurysmal dilatation in this animal model are not understood, but we hypothesize that progres- sive elastin degradation leads to loss of elastic recoil under normal blood pressure, which slowly leads to an increase in measured external diameter. This arterial expansion was accompanied by increased thickness of the media layer in the saline controls. Because these parameters were significantly reduced in PGG-treated aortas, we are tempted to speculate that direct interaction of PGG with elastin fibers preserves their integrity and helps to maintain mechanical properties of aneurysmal aortas. More detailed studies are categorically needed to better understand the mechanisms of AAA and the interaction of PGG with components of diseased aortas. Aneurysmal aorta consists of both intact and degraded elastin fibers. For potential clinical applications, we provide supportive evidence that PGG is capable of preventing degeneration of intact elastin fibers. This may account for the prevention of AAA formation in experiment 1 and the lack of AAA progression in experiment 2. However, it is not known whether PGG can interact with partially degraded elastin fibers and stabilize them against further degeneration. Advanced stages of AAA also involve collagen degen- eration, which in turn may contribute to fatal ruptures in terminal stages of AAA.35 Although polyphenolic tannin binding to collagen has been demonstrated before,36 it is not known at this point whether PGG also binds to aortic collagen in vivo and may possibly influence the outcome of developing AAA, especially in regard to preventing late AAA ruptures. These issues are currently under investiga- tion in our laboratory. In our studies, an acute application of PGG to rat aortas did not directly interfere with model-related pathogenesis; thus, PGG-mediated inhibi- tion of AAA development and progression possibly is due to direct stabilization of elastin rather than to inhibition of enzyme activities. Conclusions Maintaining the integrity of the aortic wall is vital for the prevention of AAA development. Acute localized periad- ventitial delivery of noncytotoxic concentrations of PGG inhibits elastin degeneration, attenuates aneurysmal diam- eter expansion, and hinders development of AAA in an established animal model. PGG binds strongly and specif- ically to arterial elastin, and in doing so, it preserves elastic laminar integrity and architecture despite the presence of high levels of MMPs derived from inflammatory cells. 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Cross- linking of dermal sheep collagen with tannic acid. Biomaterials. 1997; 18:749 –754. CLINICAL PERSPECTIVE Aortic aneurysms (AAs) are degenerative diseases characterized by destruction of arterial architecture and subsequent dilatation that may eventually lead to fatal ruptures. Aneurysms grow over a period of years and pose great health risks as a result of the potential to rupture, which can be fatal in �80% of cases. AAs are a serious health concern for the aging population, among the top 10 causes of death for patients �50 years of age. Currently, the sole treatment of AA is surgical replacement of the diseased artery or endovascular stent graft repair. However, reported postoperative survival rates can drop to only 50% at 10 years. Nonsurgical treatment of aneurysms does not currently exist; thus, a dire need exists for an evidence-based approach to stop this pathological process. The onset and progression of AAs are associated with elastin degradation by proteinases, which, in turn, are derived from activated vascular cells and infiltrating inflammatory cells. Using an aortic injury model that mimics human AA pathology, we show here that periarterial delivery of noncytotoxic concentrations of pentagalloyl glucose, a pharmacological agent capable of stabilizing elastin, significantly reduces AA development and controls diameter expansion by limitation of tissue degeneration. Pentagalloyl glucose delivery provides prospects for the development of clinically applicable therapies as an adjunct or stand-alone procedure that could temper development of a life-threatening pathology and thus affect thousands of patients. Isenburg et al Aneurysm Treatment by Elastin Stabilization 1737 D ow nloaded from http://ahajournals.org by on A pril 5, 2021