Characterization of focused ultrasound blood-brain barrier disruption effect on inflammation as a function of treatment parameters.

The technology of focused ultrasound-mediated disruption of the blood-brain barrier (FUS- BBB opening) has now been used in over 20 Phase 1 clinical trials to validate the safety and feasibility of BBB opening for drug delivery in patients with brain tumors and neurodegenerative diseases. The primary treatment parameters, FUS intensity and microbubble dose, are chosen to balance sufficient BBB disruption to achieve drug delivery against potential acute vessel damage leading to microhemorrhage. This can largely be achieved based on both empirical results from animal studies and by monitoring the microbubble cavitation signal in real time during the treatment. However, other safety considerations due to second order effects caused by BBB disruption, such as inflammation and alteration of neurovascular function, are not as easily measurable, may take longer to manifest and are only beginning to be understood. This study builds on previous work that has investigated the inflammatory response following FUS-BBB opening. In this study, we characterize the effect of FUS intensity and microbubble dose on the extent of BBB disruption, observed level of microhemorrhage, and degree of inflammatory response at three acute post-treatment time points in the wild-type mouse brain. Additionally, we evaluate differences related to biological sex, presence and degree of the anti- inflammatory response that develops to restore homeostasis in the brain environment, and the impact of multiple FUS-BBB opening treatments on this inflammatory response.

Extended 78% Hepatectomy in a Mouse Surgical Model.

Partial 2/3 hepatectomy in mice is used in research to study the liver's regenerative capacity and explore outcomes of liver resection in a number of disease models. In the classical partial 2/3 hepatectomy in mice, two of the five liver lobes, namely the left and median lobes representing approximately 66% of the liver mass, are resected en bloc with an expected postoperative survival of 100%. More aggressive partial hepatectomies are technically more challenging and hence, have seldom been used in mice. Our group has developed a mouse model of an extended hepatectomy technique in which three of the five liver lobes, including the left, median, and right upper lobes, are resected separately to remove approximately 78% of the total liver mass. This extended resection, in otherwise healthy mice, leaves a remnant liver that cannot always sustain adequate and timely regeneration. Failure to regenerate ultimately results in 50% postoperative lethality within 1 week due to fulminant hepatic failure. This procedure of extended 78% hepatectomy in mice represents a unique surgical model for the study of small-for-size syndrome and the evaluation of therapeutic strategies to improve liver regeneration and outcomes in the setting of liver transplantation or extended liver resection for cancer.

A20/TNFAIP3 Increases ENOS Expression in an ERK5/KLF2-Dependent Manner to Support Endothelial Cell Health in the Face of Inflammation.

Rationale: Decreased expression and activity of endothelial nitric oxide synthase (eNOS) in response to inflammatory and metabolic insults is the hallmark of endothelial cell (EC) dysfunction that preludes the development of atherosclerosis and hypertension. We previously reported the atheroprotective properties of the ubiquitin-editing and anti-inflammatory protein A20, also known as TNFAIP3, in part through interrupting nuclear factor-kappa B (NF-κB) and interferon signaling in EC and protecting these cells from apoptosis. However, A20's effect on eNOS expression and function remains unknown. In this study, we evaluated the impact of A20 overexpression or knockdown on eNOS expression in EC, at baseline and after tumor necrosis factor (TNF) treatment, used to mimic inflammation. Methods and Results: A20 overexpression in human coronary artery EC (HCAEC) significantly increased basal eNOS mRNA (qPCR) and protein (western blot) levels and prevented their downregulation by TNF. Conversely, siRNA-induced A20 knockdown decreased eNOS mRNA levels, identifying A20 as a physiologic regulator of eNOS expression. By reporter assays, using deletion and point mutants of the human eNOS promoter, and knockdown of eNOS transcriptional regulators, we demonstrated that A20-mediated increase of eNOS was transcriptional and relied on increased expression of the transcription factor Krüppel-like factor (KLF2), and upstream of KLF2, on activation of extracellular signal-regulated kinase 5 (ERK5). Accordingly, ERK5 knockdown or inhibition significantly abrogated A20's ability to increase KLF2 and eNOS expression. In addition, A20 overexpression in HCAEC increased eNOS phosphorylation at Ser-1177, which is key for the function of this enzyme. Conclusions: This is the first report demonstrating that overexpression of A20 in EC increases eNOS transcription in an ERK5/KLF2-dependent manner and promotes eNOS activating phosphorylation. This effect withstands eNOS downregulation by TNF, preventing EC dysfunction in the face of inflammation. This novel function of A20 further qualifies its therapeutic promise to prevent/treat atherosclerosis.

Embryonic periventricular endothelial cells demonstrate a unique pro-neurodevelopment and anti-inflammatory gene signature.

Brain embryonic periventricular endothelial cells (PVEC) crosstalk with neural progenitor cells (NPC) promoting mutual proliferation, formation of tubular-like structures in the former and maintenance of stemness in the latter. To better characterize this interaction, we conducted a comparative transcriptome analysis of mouse PVEC vs. adult brain endothelial cells (ABEC) in mono-culture or NPC co-culture. We identified > 6000 differentially expressed genes (DEG), regardless of culture condition. PVEC exhibited a 30-fold greater response to NPC than ABEC (411 vs. 13 DEG). Gene Ontology (GO) analysis of DEG that were higher or lower in PVEC vs. ABEC identified "Nervous system development" and "Response to Stress" as the top significantly different biological process, respectively. Enrichment in canonical pathways included HIF1A, FGF/stemness, WNT signaling, interferon signaling and complement. Solute carriers (SLC) and ABC transporters represented an important subset of DEG, underscoring PVEC's implication in blood-brain barrier formation and maintenance of nutrient-rich/non-toxic environment. Our work characterizes the gene signature of PVEC and their important partnership with NPC, underpinning their unique role in maintaining a healthy neurovascular niche, and in supporting brain development. This information may pave the way for additional studies to explore their therapeutic potential in neuro-degenerative diseases, such as Alzheimer's and Parkinson's disease.

Secondary effects on brain physiology caused by focused ultrasound-mediated disruption of the blood-brain barrier.

Focused ultrasound (FUS) combined with microbubbles is a non-invasive method for targeted, reversible disruption of the blood-brain barrier (FUS-BBB opening). This approach holds great promise for improving delivery of therapeutics to the brain. In order to achieve this clinically important goal, the approach necessarily breaks a protective barrier, temporarily, which plays a fundamental role in maintaining a homeostatic environment in the brain. Preclinical and clinical research has identified a set of treatment parameters under which this can be performed safely, whereby the BBB is disrupted to the point of being permeable to normally non-penetrant agents without causing significant acute damage to endothelial or neuronal cells. Much of the early work in this field focused on engineering questions around how to achieve optimal delivery of therapeutics via BBB disruption. However, there is increasing interest in addressing biological questions related to whether and how various aspects of neurophysiology might be affected when this fundamental protective barrier is compromised by the specific mechanisms of FUS-BBB opening. Improving our understanding of these secondary effects is becoming vital now that FUS-BBB opening treatments have entered clinical trials. Such information would help to safely expand FUS-BBB opening protocols into a wider range of drug delivery applications and may even lead to new types of treatments. In this paper, we will critically review our current knowledge of the secondary effects caused by FUS-BBB opening on brain physiology, identify areas that remain understudied, and discuss how a better understanding of these processes can be used to safely advance FUS-BBB opening into a wider range of clinical applications.