Background-We hypothesized that molecular imaging with contrast-enhanced ultrasound (CEU) and microbubbles targeted to endothelial integrins could be used to noninvasively assess early angiogenic responses to ischemia and growth factor therapy. Methods and Results-Hindlimb ischemia was produced in 48 rats by ligation of an iliac artery. Half of the animals received intramuscular sustained-release fibroblast growth factor-2 (FGF-2). Immediately after ligation and at subsequent intervals from 4 to 28 days, blood flow and oxygen tension in the proximal adductor muscles were measured by CEU perfusion imaging and phosphor quenching, respectively. Targeted CEU imaging of ␣ v -and ␣ 5 ␤ 1 -integrin expression was performed with microbubbles bearing the disintegrin echistatin. Iliac artery ligation produced a 65% to 70% reduction in blood flow and oxygen tension. In untreated ischemic muscle, muscle flow and oxygen tension partially recovered by days 14 to 28. In these animals, signal from integrin-targeted microbubbles was intense and peaked before flow increase (days 4 to 7). In comparison to untreated animals, FGF-2-treated muscle had a greater rate and extent of blood flow recovery and greater signal intensity from integrin-targeted microbubbles, which peaked before maximal recovery of flow. On immunohistology, arteriolar but not capillary density increased in the ischemic limb after ligation, the rate and degree of which were greater in FGF-2-treated rats. Immunofluorescence demonstrated intense staining for ␣ v in arterioles, the temporal course of which correlated with targeted imaging. Conclusions-Targeted CEU can be used to assess endogenous and therapeutic arteriogenesis before recovery of tissue perfusion. These results suggest that molecular imaging of integrin expression may be useful for evaluating proangiogenic therapies. (Circulation. 2005;111:3248-3254.)

BackgroundMicroglia play critical roles in the brain during homeostasis and pathological conditions. Understanding the molecular events underpinning microglial functions and activation states will further enable us to target these cells for the treatment of neurological disorders. The transcription factor PU.1 is critical in the development of myeloid cells and a major regulator of microglial gene expression. In the brain, PU.1 is specifically expressed in microglia and recent evidence from genome-wide association studies suggests that reductions in PU.1 contribute to a delayed onset of Alzheimer’s disease (AD), possibly through limiting neuroinflammatory responses.MethodsTo investigate how PU.1 contributes to immune activation in human microglia, microarray analysis was performed on primary human mixed glial cultures subjected to siRNA-mediated knockdown of PU.1. Microarray hits were confirmed by qRT-PCR and immunocytochemistry in both mixed glial cultures and isolated microglia following PU.1 knockdown. To identify attenuators of PU.1 expression in microglia, high throughput drug screening was undertaken using a compound library containing FDA-approved drugs. NanoString and immunohistochemistry was utilised to investigate the expression of PU.1 itself and PU.1-regulated mediators in primary human brain tissue derived from neurologically normal and clinically and pathologically confirmed cases of AD.ResultsBioinformatic analysis of gene expression upon PU.1 silencing in mixed glial cultures revealed a network of modified AD-associated microglial genes involved in the innate and adaptive immune systems, particularly those involved in antigen presentation and phagocytosis. These gene changes were confirmed using isolated microglial cultures. Utilising high throughput screening of FDA-approved compounds in mixed glial cultures we identified the histone deacetylase inhibitor vorinostat as an effective attenuator of PU.1 expression in human microglia. Further characterisation of vorinostat in isolated microglial cultures revealed gene and protein changes partially recapitulating those seen following siRNA-mediated PU.1 knockdown. Lastly, we demonstrate that several of these PU.1-regulated genes are expressed by microglia in the human AD brain in situ.ConclusionsCollectively, these results suggest that attenuating PU.1 may be a valid therapeutic approach to limit microglial-mediated inflammatory responses in AD and demonstrate utility of vorinostat for this purpose.Electronic supplementary materialThe online version of this article (10.1186/s13024-018-0277-1) contains supplementary material, which is available to authorized users.

BackgroundPericytes and endothelial cells are critical cellular components of the blood-brain barrier (BBB) and play an important role in neuroinflammation. To date, the majority of inflammation-related studies in endothelia and pericytes have been carried out using immortalised cell lines or non-human-derived cells. Whether these are representative of primary human cells is unclear and systematic comparisons of the inflammatory responses of primary human brain-derived pericytes and endothelia has yet to be performed.MethodsTo study the effects of neuroinflammation at the BBB, primary brain endothelial cells and pericytes were isolated from human biopsy tissue. Culture purity was examined using qPCR and immunocytochemistry. Electrical cell-substrate impedance sensing (ECIS) was used to determine the barrier properties of endothelial and pericyte cultures. Using immunocytochemistry, cytometric bead array, and ECIS, we compared the responses of endothelia and pericytes to a panel of inflammatory stimuli (IL-1β, TNFα, LPS, IFN-γ, TGF-β1, IL-6, and IL-4). Secretome analysis was performed to identify unique secretions of endothelia and pericytes in response to IL-1β.ResultsEndothelial cells were pure, moderately proliferative, retained the expression of BBB-related junctional proteins and transporters, and generated robust TEER. Both endothelia and pericytes have the same pattern of transcription factor activation in response to inflammatory stimuli but respond differently at the secretion level. Secretome analysis confirmed that endothelia and pericytes have overlapping but distinct secretome profiles in response to IL-1β. We identified several cell-type specific responses, including G-CSF and GM-CSF (endothelial-specific), and IGFBP2 and IGFBP3 (pericyte-specific). Finally, we demonstrated that direct addition of IL-1β, TNFα, LPS, and IL-4 contributed to the loss of endothelial barrier integrity in vitro.ConclusionsHere, we identify important cell-type differences in the inflammatory response of brain pericytes and endothelia and provide, for the first time, a comprehensive profile of the secretions of primary human brain endothelia and pericytes which has implications for understanding how inflammation affects the cerebrovasculature.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1167-8) contains supplementary material, which is available to authorized users.