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The role of endothelial cell-specific growth factors in the vascularization of the primate peri-ovulatory follicle was examined. Experiments were designed firstly to detect expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in granulosa cells and secondly, to determine whether gonadotrophins and/or steroids regulate their expression during the peri-ovulatory interval. Granulosa cells and follicular fluid were collected from rhesus macaques undergoing ovarian stimulation before (0 h), 12, or 36 h after a bolus of ovulatory human chorionic gonadotrophin (HCG), with or without steroid ablation and progestin replacement. VEGF, Ang-1 and Ang-2 mRNA were all detected prior to the ovulatory stimulus. Whereas follicular fluid VEGF concentrations increased 6-fold (P < 0.05) between 0 and 12 h, VEGF mRNA values were unchanged and were unaffected by steroid ablation. Ang-1 mRNA decreased from 0 to 12 h (P < 0.05), followed by a 30-fold increase (P < 0.05) at 36 h, while Ang-2 mRNA values were unchanged between 0, 12 and 36 h. Steroid ablation decreased (P < 0.05) Ang-1 mRNA at 36 h, and Ang-2 mRNA at 12 h, while only Ang-1 was restored by progestin replacement. These data suggest a dynamic expression of vascular-specific growth factors in a gonadotrophin-dependent, steroid-independent (VEGF) or steroid-dependent (Ang-1) manner in granulosa cells of peri-ovulatory follicles of primates.

To determine the temporal expression of vascular growth factors during the lifespan of the primate corpus luteum, experiments were designed to detect mRNA for vascular endothelial growth factor (VEGF), angiopoietin (Ang)-1 and Ang-2 and to localize protein expression for VEGF in macaque luteal tissue during the menstrual cycle. Corpora lutea (n = 3-5/stage) were collected during the early (3-5 days post-luteinizing hormone surge), mid- (6-8 days), mid-late (10-12 days), and late (14-16 days) luteal phase and at menstruation (17-18 days). Reverse transcription-polymerase chain reaction products equated to cDNA for VEGF, Ang-1 and Ang-2 in all corpora lutea. VEGF mRNA levels increased (P: < 0.05) from early to mid-luteal phase and declined in the late luteal phase and at menstruation. Immunostaining for VEGF was detected in the cytoplasm of steroidogenic luteal cells, with the most intense staining in the early luteal phase. Ang-1 and Ang-2 mRNA expression was low in the early to mid-luteal phase but increased (P: < 0.05) at late luteal phase before declining at menstruation. These data suggest transcriptional control of VEGF, Ang-1 and Ang-2, as well as post-transcriptional control of VEGF, in macaque corpus luteum. Dynamic expression of angiogenic/angiostatic factors appears critical for development, maintenance and regression of the luteal microvasculature during the menstrual cycle.

Remarkable changes in vascular permeability and neovascularization occur within the ovulatory, luteinizing follicle. To evaluate the importance of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in periovulatory events, sequential experiments were designed in which vehicle (PBS/0.1% BSA; controls, n = 13) or a low dose (1.5 micro g; n = 4) or a high dose (7.5 micro g; n = 4) of a VEGF antagonist, soluble VEGF receptor 1 (sVEGFR1) chimera, was injected directly into the preovulatory follicle of rhesus monkeys the day before (Day -1) or the day of (Day 0) the midcycle LH surge during spontaneous menstrual cycles. After vehicle injection, animals typically exhibited patterns and levels of serum progesterone (P(4)) that were comparable to those of untreated animals in our colony. Following low-dose sVEGFR1 injection, serum P(4) levels were diminished in two of four animals from the early to midluteal phase, but were similar to vehicle controls thereafter. In contrast, high-dose sVEGFR1 injection decreased serum P(4) levels throughout the luteal phase compared with levels in controls (P < 0.05), but it did not cause premature menstruation. Control follicles displayed indices of rupture (protruding stigmata) and luteinization. However, sVEGFR1-injected follicles exhibited signs of distension (torn surface epithelium/tunica albuginea) and luteinization, but not necessarily timely ovulation. Histological evaluation of serial sections from ovaries removed on Day 3 after treatment revealed that all (n = 3) vehicle-injected follicles ovulated, whereas half (n = 3 of 6) the sVEGFR1-injected follicles failed to ovulate and still contained an oocyte in the antrum. No appreciable differences were apparent between treatment groups in numbers of cells in luteal tissue (Day 3 or 6 after treatment) that stained positive for immunochemical or histochemical markers of proliferative (Ki67), endothelial (platelet endothelial cell adhesion molecule 1), and steroidogenic (3beta-hydroxysteroid dehydrogenase) cells. However, there was a dose-dependent increase (P < 0.05) in extracellular space in the corpus luteum by midluteal phase in sVEGFR1-treated animals. The data suggest that acute exposure to a VEGF antagonist can impair ovulation, and the subsequent development and functional capacity of the primate corpus luteum. The results are consistent with a critical role for VEGF in normal ovarian function during the periovulatory interval in primates.

The midcycle gonadotropin surge promotes vascular endothelial growth factor-A (VEGF-A) production by granulosa cells in the ovulatory follicle, but it is unclear whether primary regulators of VEGF secretion in other tissues, including hypoxia and growth factors, are also important in the ovary. To address these issues, granulosa cells were collected from rhesus monkeys during controlled ovarian stimulation either before (i.e., nonluteinized granulosa cells, NLGCs) or 27 hours after (i.e., luteinized granulosa cells, LGCs) administration of an ovulatory bolus of hCG, and cultured in fibronectin-coated wells containing a chemically defined media. When NLGCs were transferred to various O2 environments (20%, 5%, or 0% O2) or media containing 100 mM CoCl2, LH (100 ng/ml)-stimulated progesterone (P4) levels were markedly (P < 0.05) suppressed by 0% O2 or CoCl2. VEGF concentrations also declined (P < 0.05) in control, CoCl2, and CoCl2 + LH groups in 0% O2, although CoCl2 modestly increased (75% above control; P < 0.05) VEGF levels in 20% and 5% O2. When NLGCs were cultured in the presence of recombinant human insulin-like growth factor (IGF)-1, IGF-2, or insulin, there was a dose-dependent increase (P < 0.05) in VEGF levels on Day 1 of culture. Whereas optimal doses of IGF-1 or IGF-2 (50 ng/ml), hCG (100 ng/ml), and IGF plus hCG stimulated VEGF levels on Day 1, only the combination of IGF-1 or IGF-2 plus hCG increased VEGF above controls and sustained levels through Day 3 of culture. The synergistic effects of IGF and hCG were also evident in P4 levels, and were not due to changes in DNA content between treatment groups. LGCs produced much higher levels of P4 and VEGF, but the responses to different O2 concentrations and insulin-related factors were qualitatively similar to those of NLGCs. These results suggest that hypoxia is not a primary regulator of VEGF production in primate granulosa cells. However, IGFs may act in concert with the gonadotropin surge to promote VEGF secretion in the ovulatory, luteinizing follicle.

Chitosan acetate hemorrhage control dressings provided superior hemostasis to 48 ply gauze in high inguinal femoral arterial injuries. Chitosan-based dressings may provide prehospital treatment options for hemostasis in patients with severe hemorrhagic arterial injuries.

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