Angiogenesis within the ovarian follicle is an important component of ovulation. New capillary growth is initiated by the ovulatory surge of luteinizing hormone (LH), and angiogenesis is well underway at the time of follicle rupture. LH-stimulated follicular production of vascular growth factors has been shown to promote new capillary formation in the ovulatory follicle. The possibility that LH acts directly at ovarian endothelial cells to promote ovulatory angiogenesis has not been addressed. For these studies, ovaries containing ovulatory follicles were obtained from cynomolgus macaques and used for histological examination of ovarian vascular endothelial cells, and monkey ovarian microvascular endothelial cells (mOMECs) were enriched from ovulatory follicles for in vitro studies. mOMECs expressed LHCGR mRNA and protein, and immunostaining confirmed LHCGR protein in endothelial cells of ovulatory follicles in vivo. Human chorionic gonadotropin (hCG), a ligand for LHCGR, increased mOMEC proliferation, migration, and capillary-like sprout formation in vitro. Treatment of mOMECs with hCG increased cAMP, a common intracellular signal generated by LHCGR activation. The cAMP analog dibutyryl cAMP increased mOMEC proliferation in the absence of hCG. Both the protein kinase A (PKA) inhibitor H89 and the phospholipase C (PLC) inhibitor U73122 blocked hCG-stimulated mOMEC proliferation, suggesting that multiple G-proteins may mediate LHCGR action. Human ovarian microvascular endothelial cells (hOMECs) enriched from ovarian aspirates obtained from healthy oocyte donors also expressed LHCGR. hOMECs also migrated and proliferated in response to hCG. Overall, these findings indicate that the LH surge may directly activate ovarian endothelial cells to stimulate angiogenesis of the ovulatory follicle.

Staphylococcus aureus employs a multitude of immune-evasive tactics to circumvent host defenses including the complement system, a component of innate immunity central to controlling bacterial infections. With antibiotic resistance becoming increasingly common, there is a dire need for novel therapies. Previously, we have shown that S. aureus binds the complement regulator factor H (FH) via surface protein SdrE to inhibit complement. To address the need for novel therapeutics and take advantage of the FH:SdrE interaction, we examined the effect of a fusion protein comprised of the SdrE-interacting domain of FH coupled with IgG Fc on complement-mediated opsonophagocytosis and bacterial killing of community associated methicillin-resistant S. aureus. S. aureus bound significantly more FH-Fc compared to Fc-control proteins and FH-Fc competed with serum FH for S. aureus binding. FH-Fc treatment increased C3-fragment opsonization of S. aureus for both C3b and iC3b, and boosted generation of the anaphylatoxin C5a. In 5 and 10% serum, FH-Fc treatment significantly increased S. aureus killing by polymorphonuclear cells. This anti-staphylococcal effect was evident in 75% (3/4) of clinical isolates tested. This study demonstrates that FH-Fc fusion proteins have the potential to mitigate the protective effects of bound serum FH rendering S. aureus more vulnerable to the host immune system. Thus, we report the promise of virulence-factor-targeted fusion-proteins as an avenue for prospective anti-staphylococcal therapeutic development.

Staphylococcus aureus is a significant human pathogen that causes a multitude of superficial and invasive infections worldwide. As antibiotic resistance is increasing, novel therapies are in dire need. As a master of immune evasion, S. aureus produces several virulence factors to persist in the host. A primary target of staphylococcal immune evasion is the Complement system, a component of innate immunity central to controlling bacterial infections. We have previously shown that S. aureus binds Complement regulator Factor H (FH) via surface protein SdrE to inhibit Complement. To address the need for novel therapeutics and take advantage of the SdrE:FH interaction, we tested the binding and downstream effect of a fusion protein comprised of the SdrE-interacting domain of FH coupled with IgG1 Fc using a S. aureus clinical isolate known to bind FH. S. aureus bound significantly more FH-Fc than Fc-control proteins indicating a greater affinity for SdrE compared to S. aureus Fc-binding proteins Protein A and Sbi. In the presence of normal human serum, FH-Fc competitively bound to S. aureus and reduced S. aureus recruitment of serum FH compared to Fc-control. When examining the effect of FH-Fc on complement-mediated opsonization of S. aureus, treatment with FH-Fc resulted in a slight increase in C3-fragment deposition and a significant increase in C5a generation compared to control. This may be attributed to the cumulative effect of serum FH displacement plus a boost in classical pathway activation via additional Fc presence afforded by SdrE:FH-Fc binding. Taken together, these data support FH-Fc as a potential anti-staphylococcal therapeutic. Future studies will explore how FH-Fc affects S. aureus survival when challenged with phagocytes.