Ciliogenesis mechanisms mediated by PAK2-ARL13B signaling in brain endothelial cells is responsible for vascular stability.

In the developing vasculature, cilia, microtubule-based organelles that project from the apical surface of endothelial cells (ECs), have been identified to function cell autonomously to promote vascular integrity and prevent hemorrhage. To date, the underlying mechanisms of endothelial cilia formation (ciliogenesis) are not fully understood. Understanding these mechanisms is likely to open new avenues for targeting EC-cilia to promote vascular stability. Here, we hypothesized that brain ECs ciliogenesis and the underlying mechanisms that control this process are critical for brain vascular stability. To investigate this hypothesis, we utilized multiple approaches including developmental zebrafish model system and primary cell culture systems. In the p21 activated kinase 2 (pak2a) zebrafish vascular stability mutant [redhead (rhd)] that shows cerebral hemorrhage, we observed significant decrease in cilia-inducing protein ADP Ribosylation Factor Like GTPase 13B (Arl13b), and a 4-fold decrease in cilia numbers. Overexpressing ARL13B-GFP fusion mRNA rescues the cilia numbers (1-2-fold) in brain vessels, and the cerebral hemorrhage phenotype. Further, this phenotypic rescue occurs at a critical time in development (24 h post fertilization), prior to initiation of blood flow to the brain vessels. Extensive biochemical mechanistic studies in primary human brain microvascular ECs implicate ligands platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor-A (VEGF-A) trigger PAK2-ARL13B ciliogenesis and signal through cell surface VEGFR-2 receptor. Thus, collectively, we have implicated a critical brain ECs ciliogenesis signal that converges on PAK2-ARL13B proteins to promote vascular stability.

Chemokine CCL28 Is a Potent Therapeutic Agent for Oropharyngeal Candidiasis.

Candida albicans is a commensal organism that causes life-threatening or life-altering opportunistic infections. Treatment of Candida infections is limited by the paucity of antifungal drug classes. Naturally occurring antimicrobial peptides are promising agents for drug development. CCL28 is a CC chemokine that is abundant in saliva and has in vitro antimicrobial activity. In this study, we examine the in vivo Candida killing capacity of CCL28 in oropharyngeal candidiasis as well as the spectrum and mechanism of anti-Candida activity. In the mouse model of oropharyngeal candidiasis, application of wild-type CCL28 reduces oral fungal burden in severely immunodeficient mice without causing excessive inflammation or altering tissue neutrophil recruitment. CCL28 is effective against multiple clinical strains of C. albicans Polyamine protein transporters are not required for CCL28 anti-Candida activity. Both structured and unstructured CCL28 proteins show rapid and sustained fungicidal activity that is superior to that of clinical antifungal agents. Application of wild-type CCL28 to C. albicans results in membrane disruption as measured by solute movement, enzyme leakage, and induction of negative Gaussian curvature on model membranes. Membrane disruption is reduced in CCL28 lacking the functional C-terminal tail. Our results strongly suggest that CCL28 can exert antifungal activity in part via membrane permeation and has potential for development as an anti-Candida therapeutic agent without inflammatory side effects.