title: Interanimal variability in preexisting collaterals is a major factor determining outcome in experimental angiogenesis trials.
abstract: Despite numerous animal trials reporting that cell therapy promotes collateral flow, clinical trials have not convincingly shown benefit. Patient-related risk factors are often used to explain these discrepancies. However, during the course of our own angiogenesis studies using mice, we noted large anatomical variability in collateral vessels. The purpose of the present investigation was to define how important this factor might be in determining intervention outcomes. Hindlimb ischemia was induced in BALB/c mice by ligating the superficial femoral artery. After 24 h, animals were treated by injecting the adductor muscle with either control media or cultured mesenchymal stem cells (MSCs). Blood flow recovery was measured using laser-Doppler [laser-Doppler perfusion imaging (LDPI) ratio]. In a second experiment, mice were stratified 24 h after arterial ligation before treatment by using a simple clinical score of the ligated leg: 1, able to flex, mild discoloration; 2, no flexion, mild discoloration; 3, severe discoloration; and 4, any necrosis. Without stratification, blood flow recovery significantly increased in the MSC-treated group (P < 0.05, n = 6 MSC group, n = 7 media group). In the experiment employing stratification, all differences between the groups disappeared (n = 11 MSC group, n = 10 media group; P = 0.3). Furthermore, we found a striking inverse correlation between clinical score on day 1 and the LDPI ratio on day 28 (P < 0.0001; n = 79). Anatomical confirmation of the disparity in preexisting collaterals was found in two different mouse strains using microscopic computed tomography. In conclusion, there is substantial interanimal variability in preexisting collateral flow, and this variability can importantly influence outcome. To overcome this, either animals must be stratified before treatment, the number of animals must be increased substantially, or, preferably, both.
authors.affiliation: Cardiovascular Research Institute, MedStar Research Institute, Washington Hospital Center, Washington, District of Columbia 20010, USA. stephan.zbinden@insel.ch
authors.forename: Stephan
authors.initials: S
authors.lastname: Zbinden
authors.forename: Leonardo C
authors.initials: LC
authors.lastname: Clavijo
authors.forename: Birgit
authors.initials: B
authors.lastname: Kantor
authors.forename: Hakim
authors.initials: H
authors.lastname: Morsli
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Cortes
authors.forename: James A
authors.initials: JA
authors.lastname: Andrews
authors.forename: Gil Jin
authors.initials: GJ
authors.lastname: Jang
authors.forename: Mary Susan
authors.initials: MS
authors.lastname: Burnett
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Epstein
mesh_terms: D000818:Animals; D003097:Collateral Circulation / Q000502:physiology*; D016319:Corrosion Casting; D004195:Disease Models, Animal; D005263:Femoral Artery; D006614:Hindlimb / Q000098:blood supply; D007511:Ischemia / Q000473:pathology / Q000503:physiopathology* / Q000628:therapy*; D017078:Laser-Doppler Flowmetry; D008026:Ligation; D045164:Mesenchymal Stem Cell Transplantation; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009336:Necrosis; D011237:Predictive Value of Tests; D012039:Regional Blood Flow; D012680:Sensitivity and Specificity