Initiating angiotensin II at lower vasopressor doses in vasodilatory shock: an exploratory post-hoc analysis of the ATHOS-3 clinical trial.

BACKGROUND: High dose vasopressors portend poor outcome in vasodilatory shock. We aimed to evaluate the impact of baseline vasopressor dose on outcomes in patients treated with angiotensin II (AT II). METHODS: Exploratory post-hoc analysis of the Angiotensin II for the Treatment of High-Output Shock (ATHOS-3) trial data. The ATHOS-3 trial randomized 321 patients with vasodilatory shock, who remained hypotensive (mean arterial pressure of 55-70 mmHg) despite receiving standard of care vasopressor support at a norepinephrine-equivalent dose (NED) > 0.2 µg/kg/min, to receive AT II or placebo, both in addition to standard of care vasopressors. Patients were grouped into low (≤ 0.25 µg/kg/min; n = 104) or high (> 0.25 µg/kg/min; n = 217) NED at the time of study drug initiation. The primary outcome was the difference in 28-day survival between the AT II and placebo subgroups in those with a baseline NED ≤ 0.25 µg/kg/min at the time of study drug initiation. RESULTS: Of 321 patients, the median baseline NED in the low-NED subgroup was similar in the AT II (n = 56) and placebo (n = 48) groups (median of each arm 0.21 µg/kg/min, p = 0.45). In the high-NED subgroup, the median baseline NEDs were also similar (0.47 µg/kg/min AT II group, n = 107 vs. 0.45 µg/kg/min placebo group, n = 110, p = 0.75). After adjusting for severity of illness, those randomized to AT II in the low-NED subgroup were half as likely to die at 28-days compared to placebo (HR 0.509; 95% CI 0.274-0.945, p = 0.03). No differences in 28-day survival between AT II and placebo groups were found in the high-NED subgroup (HR 0.933; 95% CI 0.644-1.350, p = 0.71). Serious adverse events were less frequent in the low-NED AT II subgroup compared to the placebo low-NED subgroup, though differences were not statistically significant, and were comparable in the high-NED subgroups. CONCLUSIONS: This exploratory post-hoc analysis of phase 3 clinical trial data suggests a potential benefit of AT II introduction at lower doses of other vasopressor agents. These data may inform design of a prospective trial. TRIAL REGISTRATION: The ATHOS-3 trial was registered in the clinicaltrials.gov repository (no. NCT02338843). Registered 14 January 2015.

BAX regulates follicular endowment in mice.

It is believed that the endowment of primordial follicles in mammalian ovaries is finite. Once follicles are depleted, infertility ensues. Thus, the size of the initial endowment has consequences for fertility and reproductive longevity. Follicular endowment is comprised of various processes that culminate with the incorporation of meiosis-arrested oocytes into primordial follicles. Apoptosis is prominent during follicular endowment, and apoptosis regulatory genes are involved in its regulation. Conflicting data exist with regard to the role of the proapoptotic Bcl-2 associated X protein (BAX) in follicular endowment. Therefore, we investigated the role of BAX during follicular endowment in embryonic and neonatal ovaries. We found that BAX is involved in regulating follicular endowment in mice. Deletion of Bax yields increased oocyte numbers in embryonic ovaries and increased follicle numbers in neonatal ovaries when compared with wild-type ovaries. Increased follicular endowment in Bax -/- ovaries is not due to enhanced germ cell viability. Further, it is not due to an increased primordial germ cell (PGC) allotment, a delay in the onset of meiosis, or altered proliferative activity of oogonia. Instead, our data suggest that the regulatory activity of BAX in follicular endowment likely occurs during PGC migration, prior to PGC colonization of the gonad.

BAX is involved in regulating follicular growth, but is dispensable for follicle atresia in adult mouse ovaries.

Mammalian females are endowed with a finite number of primordial follicles at birth or shortly thereafter. Immediately following the formation of the primordial follicle pool, cohorts of these follicles are recruited to begin growth, and this recruitment continues until the primordial follicle population is depleted. Once recruited, a follicle will either grow and ovulate or undergo atresia. Follicle atresia results from the apoptotic death of follicular cells. Members of the BCL-2 family of proteins are important regulators of apoptosis in most cells including in the ovary. Here, we tested the hypothesis that the proapoptotic BAX is an important regulator of follicle survival. We used a variety of histological and biochemical techniques to investigate the impact of Bax deletion on follicle growth and death. We observed that the Bax deletion results in delayed vaginal opening and altered follicular growth. Young adult Bax-deficient ovaries contained increased numbers of primordial follicles and a trend towards reduced numbers of growing follicles. Bax deficiency led to a reduction in average litter size, and also a reduction in the number of oocytes ovulated in response to exogenous gonadotropins. In contrast, Bax deficiency did not alter follicle atresia. In conclusion, BAX appears to be an important regulator of follicle growth, but is dispensable for follicle atresia in mice.

Tumor necrosis factor (TNF) receptor type 2 is an important mediator of TNF alpha function in the mouse ovary.

It is believed that a finite pool of primordial follicles is established during embryonic and neonatal life. At birth, the mouse ovary consists of clusters of interconnected oocytes surrounded by pregranulosa cells. Shortly after birth these structures, termed germ cell cysts or nests (GCN), break down to facilitate primordial follicle formation. Tumor necrosis factor alpha (TNF) is a widely expressed protein with myriad functions. TNF is expressed in the ovary and may regulate GCN breakdown in rats. We investigated whether it participates in GCN breakdown and follicle formation in mice by using an in vitro ovary culture system as well as mutant animal models. We found that TNF and both receptors (TNFRSF1A and TNFRSF1B) are expressed in neonatal mouse ovaries and that TNF promotes oocyte death in neonatal ovaries in vitro. However, deletion of either receptor did not affect follicle endowment, suggesting that TNF does not regulate GCN breakdown in vivo. Tnfrsf1b deletion led to an apparent acceleration of follicular growth and a concomitant expansion of the primordial follicle population. This expansion of the primordial follicle population does not appear to be due to decreased primordial follicle atresia, although this cannot be ruled out completely. This study demonstrates that mouse oocytes express both TNF receptors and are sensitive to TNF-induced death. Additionally, TNFRSF1B is demonstrated to be an important mediator of TNF function in the mouse ovary and an important regulator of folliculogenesis.

Methoxychlor directly affects ovarian antral follicle growth and atresia through Bcl-2- and Bax-mediated pathways.

Methoxychlor (MXC) is an organochlorine pesticide and reproductive toxicant. While in vivo studies indicate that MXC exposure increases antral follicle atresia, in part by altering apoptotic regulators (Bcl-2 and Bax), they do not distinguish whether MXC does so via direct or indirect mechanisms. Therefore, we utilized an in vitro follicle culture system to test the hypothesis that MXC is directly toxic to antral follicles, and that overexpression of anti-apoptotic Bcl-2, or deletion of pro-apoptotic Bax, protects antral follicles from MXC-induced toxicity. Antral follicles were isolated from wild-type (WT), Bcl-2 overexpressing (Bcl-2 OE), or Bax deficient (BaxKO) mice, and exposed to dimethylsulfoxide (control) or MXC (1-100 microg/ml) for 96 h. Follicle diameters were measured every 24 h to assess growth. After 96 h, follicles were histologically evaluated for atresia or collected for quantitative PCR analysis of Bcl-2 and Bax mRNA levels. MXC (10-100 microg/ml) significantly inhibited antral follicle growth at 72 and 96 h, and increased atresia (100 microg/ml) compared to controls at 96 h. Furthermore, MXC increased Bax mRNA levels between 48-96 h and decreased Bcl-2 mRNA levels at 96 h. While MXC inhibited growth of WT antral follicles beginning at 72 h, it did not inhibit growth of Bcl-2 OE or BaxKO follicles until 96 h. MXC also increased atresia of small and large WT and BaxKO antral follicles over controls, but it did not increase atresia of large Bcl-2 OE antral follicles over controls. These data suggest that MXC directly inhibits follicle growth partly by Bcl-2 and Bax pathways, and increases atresia partly through Bcl-2 pathways.