Genetic loci that control vascular endothelial growth factor-induced angiogenesis.

Angiogenesis is regulated by the balance between angiogenic stimulators and inhibitors. Numerous reports have demonstrated that tumors induce aggressive angiogenesis by up-regulating the production of angiogenesis stimulating growth factors to overcome the baseline levels of endogenous inhibitors. However, the possibility of large differences in the host's responsiveness to angiogenic factors has been largely overlooked. Using the corneal micropocket neovascularization assay, we have observed >10-fold differences in responsiveness to either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) among various mouse strains. The inheritance pattern observed for these traits supported a QTL (quantitative trait locus) approach to mapping the genes responsible for the differences in angiogenic responsiveness. To overcome variability in the assay, we used recombinant inbred lines to map this phenotype. In the BXD series of recombinant inbred mouse strains, we have mapped the regions responsible for regulating VEGF-induced angiogenesis using both composite interval mapping and multiple interval mapping. Both approaches link VEGF responsiveness to regions on chromosomes 2 (near D2Mit6) and 10 (near D10Mit20). Candidate angiogenesis-related genes in these regions include those for collagen XVIII/endostatin, matrix metalloproteinase 11, integrin beta2, prostaglandin D2 synthase, and interleukin-1 receptor antagonist.

Genetic loci that control the angiogenic response to basic fibroblast growth factor.

Angiogenesis is controlled by a balance between stimulatory growth factors and endogenous inhibitors. We propose that the balance of stimulators and inhibitors, as well as the general sensitivity of the endothelium to these factors, varies from individual to individual. Indeed, we have found that individual mouse strains have dramatically different responses to growth factor-induced neovascularization. Quantitative trait loci (QTLs), which influence the extent of angiogenesis induced by vascular endothelial growth factor (VEGF), were previously identified by our laboratory. Since genetic susceptibility may vary according to the angiogenic stimulator, we have undertaken a similar mapping approach to identify QTLs that influence basic fibroblast growth factor (FGF2) induced neovascularization in the BXD series of recombinant inbred mouse strains. Composite and multiple interval mapping identified areas of chromosomes 4, 13, 15, and 18. These new angiogenesis QTLs, named AngFq1 through AngFq4 (for angiogenesis due to FGF2), are different from previously identified VEGF QTLs. The mapped regions contain several genes involved in the angiogenic process including matrix metalloproteinase 16, eph receptor A7, angiopoetin 1, endothelial lipase, and autotaxin. Differences in these regions may influence individual susceptibility to angiogenesis related diseases such as cancer, macular degeneration, atherosclerosis, and arthritis.

Nonsteroidal antiinflammatory drugs differentially suppress endometriosis in a murine model.

OBJECTIVE: To determine whether nonsteroidal antiinflammatory drugs (NSAIDs) affect the establishment and progression of endometriotic lesions in a murine model. DESIGN: Pharmacologic intervention in a surgically induced murine model of abdominal/peritoneal endometriosis. SETTING: Animal research facility. PATIENT(S): Eight-week-old, female C57BL/6 mice. INTERVENTION(S): After implantation of autologous endometrium, mice were randomized into groups and treated with one of several NSAIDs or the vehicle-matched control for 4 weeks. MAIN OUTCOME MEASURE(S): Establishment, growth, and total burden of endometriotic lesions. RESULT(S): The NSAIDs differentially inhibited lesion establishment and growth, resulting in significantly reduced disease burden. Compared with controls (5.7 +/- 2.3 mm(2)), lesion burden was reduced by celecoxib (1.3 +/- 1.2 mm(2)), indomethacin (1.4 +/- 1.4 mm(2)), naproxen (2.7 +/- 1.2 mm(2)), sulindac (3.1 +/- 1.5 mm(2)), rofecoxib (3.4 +/- 3.0 mm(2)), and ibuprofen (4.1 +/- 1.4 mm(2)). In contrast, aspirin (5.9 +/- 1.2 mm(2)) had no statistically significant effect. Uninterrupted estrus cycling was confirmed by vaginal exams and smears in celecoxib-treated mice. CONCLUSION(S): Chronic administration of certain NSAIDs limits the progression of endometriosis in this murine model. The data suggest that NSAID selection in the treatment of endometriosis should be extended beyond pain management to maximize the inhibitory effect on disease burden.

The classical pink-eyed dilution mutation affects angiogenic responsiveness.

Angiogenesis is the process by which new blood vessels are formed from existing vessels. Mammalian populations, including humans and mice, harbor genetic variations that alter angiogenesis. Angiogenesis-regulating gene variants can result in increased susceptibility to multiple angiogenesis-dependent diseases in humans. Our efforts to dissect the complexity of the genetic diversity that regulates angiogenesis have used laboratory animals due to the availability of genome sequence for many species and the ability to perform high volume controlled breeding. Using the murine corneal micropocket assay, we have observed more than ten-fold difference in angiogenic responsiveness among various mouse strains. This degree of difference is observed with either bFGF or VEGF induced corneal neovascularization. Ongoing mapping studies have identified multiple loci that affect angiogenic responsiveness in several mouse models. In this study, we used F2 intercrosses between C57BL/6J and the 129 substrains 129P1/ReJ and 129P3/J, as well as the SJL/J strain, where we have identified new QTLs that affect angiogenic responsiveness. In the case of AngFq5, on chromosome 7, congenic animals were used to confirm the existence of this locus and subcongenic animals, combined with a haplotype-based mapping approach that identified the pink-eyed dilution mutation as a candidate polymorphism to explain AngFq5. The ability of mutations in the pink-eyed dilution gene to affect angiogenic response was demonstrated using the p-J allele at the same locus. Using this allele, we demonstrate that pink-eyed dilution mutations in Oca2 can affect both bFGF and VEGF-induced corneal angiogenesis.

Endostatin inhibits the growth of endometriotic lesions but does not affect fertility.

OBJECTIVE: To determine whether endometriosis can be treated with the angiogenesis inhibitor endostatin and the effect of this treatment on fertility and reproduction. DESIGN: Pharmacologic intervention in a surgically induced model of endometriosis and in female mice undergoing mating. SETTING: Animal research facility. ANIMAL(S): Eight-week-old, female C57BL/6 and SCID mice. INTERVENTION(S): After implantation of autologous endometrium, mice received endostatin or the vehicle-matched control for 4 weeks. For the reproductive function study, mice receiving endostatin or vehicle were mated and reproductive functions were observed. MAIN OUTCOME MEASURE(S): Growth of endometriotic lesions after 4 weeks of treatment; estrous cycling, corpus luteum formation, serum hormone levels, and mating time as fertility measures; and pregnancy rates, length of pregnancy, fetal vitality, number, and outcome of litter as reproductive measures. RESULT(S): Endostatin suppressed the growth of endometriotic lesions by 47% compared with controls. Estrous cycling and corpus luteum formation were normal in both groups. Female mice receiving endostatin were as fertile as mice receiving vehicle, had normal pregnancies, and delivered the same number of pups. The offspring were healthy without teratogenic stigmata and reproduced normally themselves. CONCLUSION(S): Antiangiogenic therapy with endostatin may present a promising novel, nontoxic therapeutic option for patients with endometriosis.

Injection of antiangiogenic agents into the macaque preovulatory follicle: disruption of corpus luteum development and function.

Ovulation and conversion of the follicle into the corpus luteum involve remarkable changes in vascular permeability and neovascularization of the luteinizing granulosa layer. To evaluate the importance of these vascular events in follicle rupture and luteal development, sequential experiments were designed in which vehicle or angiogenic inhibitors (TNP-470 or angiostatin) were injected directly into the preovulatory follicle of rhesus monkeys during spontaneous menstrual cycles. After control injections, 13 of 14 animals exhibited serum levels of progesterone (P) during the subsequent luteal phase that were comparable to untreated animals in our colony. Following low-dose (400 pg/mL) TNP-470, serum P levels increased normally until d 8 of the luteal phase, but then declined prematurely by d 9 (p < 0.05 compared to controls) and remained below controls until menses. Following high-dose (2 microg/mL) TNP-470, serum P levels were diminished in the early luteal phase (d 3-5; p < 0.05 compared to controls), but reached typical levels at mid luteal phase, only to decline prematurely by d 9 (p < 0.05) and remain low until menses. Control ovaries displayed indices of follicle rupture (protruding stigmata) and luteinization. TNP-470-treated ovaries exhibited signs of distension (torn surface epithelium/tunica albuginea) and luteinization; however, a well-formed stigmata was not observed. A "trapped" oocyte was not observed in serial sections of developing corpora lutea from control or TNP-470-treated animals. However, the early corpus luteum of TNP-470-injected ovaries contained pockets of excessive numbers of blood cells that were absent in controls. Angiostatin did not alter serum P levels or ovarian morphology compared to controls. These data suggest that acute exposure to the antiangiogenic agent TNP-470 impairs the development and functional capacity of the primate corpus luteum in a dose-dependent manner. The results are consistent with a critical role for angiogenesis in cyclic ovarian function in primates.