Therapeutic Agent Delivery Across the Blood–Brain Barrier Using Focused Ultrasound

Specialized features of vasculature in the central nervous system greatly limit therapeutic treatment options for many neuropathologies. Focused ultrasound, in combination with circulating microbubbles, can be used to transiently and noninvasively increase cerebrovascular permeability with a high level of spatial precision. For minutes to hours following sonication, drugs can be administered systemically to extravasate in the targeted brain regions and exert a therapeutic effect, after which permeability returns to baseline levels. With the wide range of therapeutic agents that can be delivered using this approach and the growing clinical need, focused ultrasound and microbubble (FUS+MB) exposure in the brain has entered human testing to assess safety. This review outlines the use of FUS+MB-mediated cerebrovascular permeability enhancement as a drug delivery technique, details several technical and biological considerations of this approach, summarizes results from the clinical trials conducted to date, and discusses the future direction of the field. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 23 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Sphingolipids affect fibrinogen-induced caveolar transcytosis and cerebrovascular permeability

Inflammation-induced vascular endothelial dysfunction can allow plasma proteins to cross the vascular wall, causing edema. Proteins may traverse the vascular wall through two main pathways, the paracellular and transcellular transport pathways. Paracellular transport involves changes in endothelial cell junction proteins, while transcellular transport involves caveolar transcytosis. Since both processes are associated with filamentous actin formation, the two pathways are interconnected. Therefore, it is difficult to differentiate the prevailing role of one or the other pathway during various pathologies causing an increase in vascular permeability. Using a newly developed dual-tracer probing method, we differentiated transcellular from paracellular transport during hyperfibrinogenemia (HFg), an increase in fibrinogen (Fg) content. Roles of cholesterol and sphingolipids in formation of functional caveolae were assessed using a cholesterol chelator, methyl-β-cyclodextrin, and the de novo sphingolipid synthesis inhibitor myriocin. Fg-induced formation of functional caveolae was defined by association and colocalization of Na+-K+-ATPase and plasmalemmal vesicle-associated protein-1 with use of Förster resonance energy transfer and total internal reflection fluorescence microscopy, respectively. HFg increased permeability of the endothelial cell layer mainly through the transcellular pathway. While MβCD blocked Fg-increased transcellular and paracellular transport, myriocin affected only transcellular transport. Less pial venular leakage of albumin was observed in myriocin-treated HFg mice. HFg induced greater formation of functional caveolae, as indicated by colocalization of Na+-K+-ATPase with plasmalemmal vesicle-associated protein-1 by Förster resonance energy transfer and total internal reflection fluorescence microscopy. Our results suggest that elevated blood levels of Fg alter cerebrovascular permeability mainly by affecting caveolae-mediated transcytosis through modulation of de novo sphingolipid synthesis.

Ablation of MMP9 Gene Ameliorates Paracellular Permeability and Fibrinogen–Amyloid Beta Complex Formation during Hyperhomocysteinemia

Increased blood level of homocysteine (Hcy), called hyperhomocysteinemia (HHcy) accompanies many cognitive disorders including Alzheimer's disease. We hypothesized that HHcy-enhanced cerebrovascular permeability occurs via activation of matrix metalloproteinase-9 (MMP9) and leads to an increased formation of fibrinogen- β-amyloid (Fg-A β) complex. Cerebrovascular permeability changes were assessed in C57BL/6J (wild type, WT), cystathionine- β-synthase heterozygote (Cbs+/−, a genetic model of HHcy), MMP9 gene knockout (Mmp9−/−), and Cbs and Mmp9 double knockout (Cbs+/−/Mmp9−/−) mice using a dual-tracer probing method. Expression of vascular endothelial cadherin (VE-cadherin) and Fg-A β complex formation was assessed in mouse brain cryosections by immunohistochemistry. Short-term memory of mice was assessed with a novel object recognition test. The cerebrovascular permeability in Cbs +/−- mice was increased via mainly the paracellular transport pathway. VE-cadherin expression was the lowest and Fg-A β complex formation was the highest along with the diminished short-term memory in Cbs +/− mice. These effects of HHcy were ameliorated in Cbs+/−/Mmp9−/− mice. Thus, HHcy causes activation of MMP9 increasing cerebrovascular permeability by downregulation of VE-cadherin resulting in an enhanced formation of Fg-A β complex that can be associated with loss of memory. These data may lead to the identification of new targets for therapeutic intervention that can modulate HHcy-induced cerebrovascular permeability and resultant pathologies.

Fibrinogen-Induced Increased Pial Venular Permeability in Mice

Elevated blood level of Fibrinogen (Fg) is commonly associated with vascular dysfunction. We tested the hypothesis that at pathologically high levels, Fg increases cerebrovascular permeability by activating matrix metalloproteinases (MMPs). Fibrinogen (4 mg/mL blood concentration) or equal volume of phosphate-buffered saline (PBS) was infused into male wild-type (WT; C57BL/6J) or MMP-9 gene knockout (MMP9-/-) mice. Pial venular leakage of fluorescein isothiocyanate-bovine serum albumin to Fg or PBS alone and to topically applied histamine (105mol/L) were assessed. Intravital fluorescence microscopy and image analysis were used to assess cerebrovascular protein leakage. Pial venular macromolecular leakage increased more after Fg infusion than after infusion of PBS in both (WT and MMP9-/-) mice but was more pronounced in WT compared with MMP9-/-mice. Expression of vascular endothelial cadherin (VE-cadherin) was less and plasmalemmal vesicle-associated protein-1 (PV-1) was greater in Fg-infused than in PBS-infused both mice groups. However, in MMP9-/-mice, VE-cadherin expression was greater and PV-1 expression was less than in WT mice. These data indicate that at higher levels, Fg compromises microvascular integrity through activation of MMP-9 and downregulation of VE-cadherin and upregulation of PV-1. Our results suggest that elevated blood level of Fg could have a significant role in cerebrovascular dysfunction and remodeling.