cytoNet: Spatiotemporal network analysis of cell communities.

We introduce cytoNet, a cloud-based tool to characterize cell populations from microscopy images. cytoNet quantifies spatial topology and functional relationships in cell communities using principles of network science. Capturing multicellular dynamics through graph features, cytoNet also evaluates the effect of cell-cell interactions on individual cell phenotypes. We demonstrate cytoNet's capabilities in four case studies: 1) characterizing the temporal dynamics of neural progenitor cell communities during neural differentiation, 2) identifying communities of pain-sensing neurons in vivo, 3) capturing the effect of cell community on endothelial cell morphology, and 4) investigating the effect of laminin α4 on perivascular niches in adipose tissue. The analytical framework introduced here can be used to study the dynamics of complex cell communities in a quantitative manner, leading to a deeper understanding of environmental effects on cellular behavior. The versatile, cloud-based format of cytoNet makes the image analysis framework accessible to researchers across domains.

Ultrasound-enhanced bevacizumab release from echogenic liposomes for inhibition of atheroma progression.

CONTEXT: Bevacizumab (BEV) is a monoclonal antibody to vascular endothelial growth factor (VEGF) that ameliorates atheroma progression by inhibiting neovascularization. OBJECTIVE: We aimed to determine whether BEV release from echogenic liposomes (BEV-ELIP) could be enhanced by color Doppler ultrasound (US) and whether the released BEV inhibits VEGF expression by endothelial cells in vitro. MATERIALS AND METHODS: BEV-ELIP samples were subjected to 6 MHz color Doppler ultrasound (MI = 0.4) for 5 min. We assessed release of BEV with a direct ELISA and with fluoresceinated BEV (FITC-BEV) loaded into ELIP by the same method. Human umbilical vein endothelial cell (HUVEC) cultures were stimulated to express VEGF by 10 nM phorbol-12-myristate 13-acetate (PMA). Cell-associated VEGF levels were determined using a cell-based ELISA. RESULTS: Overall, US caused an additional 100 µg of BEV to be released or exposed per BEV-ELIP aliquot within 60 min BEV-ELIP treated with US inhibited VEGF expression by 90% relative to non-treated controls and by 70% relative to BEV-ELIP without US. Also, US-treated BEV-ELIP inhibited HUVEC proliferation by 64% relative to untreated controls and by 45% relative to BEV-ELIP without US. DISCUSSION AND CONCLUSION: We have demonstrated that BEV-ELIP retains its VEGF-binding activity in a liposomal formulation and that clinical Doppler US can significantly increase that activity, both by releasing free BEV and by enhancing the surface exposure of the immunoreactive antibody.

Nitric oxide-loaded echogenic liposomes for treatment of vasospasm following subarachnoid hemorrhage.

Delayed cerebral vasospasm following subarachnoid hemorrhage causes severe ischemic neurologic deficits leading to permanent neurologic dysfunction or death. Reduced intravascular and perivascular nitric oxide (NO) availability is a primary pathophysiology of cerebral vasospasm. In this study, we evaluated NO-loaded echogenic liposomes (NO-ELIP) for ultrasound-facilitated NO delivery to produce vasodilation for treatment of vasospasm following subarachnoid hemorrhage. We investigated the vasodilative effects of NO released from NO-ELIP both ex vivo and in vivo. Liposomes containing phospholipids and cholesterol were prepared, and NO was encapsulated. The encapsulation and release of NO from NO-ELIP were determined by the syringe/vacuum method and ultrasound imaging. The ex vivo vasodilative effect of NO-ELIP was investigated using rabbit carotid arteries. Arterial vasodilation was clearly observed with NO-ELIP exposed to Doppler ultrasound whereas there was little vasodilative effect without exposure to Doppler ultrasound in the presence of red blood cells. Penetration of NO into the arterial wall was determined by fluorescent microscopy. The vasodilative effects of intravenously administered NO-ELIP in vivo were determined in a rat subarachnoid hemorrhage model. NO-ELIP with ultrasound activation over the carotid artery demonstrated effective arterial vasodilation in vivo resulting in improved neurologic function. This novel methodology for ultrasound-controlled delivery of NO has the potential for therapeutic treatment of vasospasm following subarachnoid hemorrhage. This ultrasound-controlled release strategy provides a new avenue for targeted bioactive gas and therapeutic delivery for improved stroke treatment.

Therapeutic time window and dose dependence of xenon delivered via echogenic liposomes for neuroprotection in stroke.

AIMS: Neurologic impairment following ischemic injury complicates the quality of life for stroke survivors. Xenon (Xe) has favorable neuroprotective properties to modify stroke. Xe delivery is hampered by a lack of suitable administration strategies. We have developed Xe-containing echogenic liposomes (Xe-ELIP) for systemic Xe delivery. We investigated the time window for Xe-ELIP therapeutic effect and the most efficacious dose for neuroprotection. Molecular mechanisms for Xe neuroprotection were investigated. METHODS: Xenon-containing echogenic liposomes were created by a previously developed pressurization-freezing method. Following right middle cerebral artery occlusion (2 h), animals were treated with Xe-ELIP at 2, 3, or 5 h to determine time window of therapeutic effect. The neuroprotectant dosage for optimal effect was evaluated 3 h after stroke onset. Expression of brain-derived neurotrophic factor (BDNF), protein kinase B (Akt), and mitogen-activated protein kinases (MAPK) was determined. RESULTS: Xenon-containing echogenic liposomes administration for up to 5 h after stroke onset reduced infract size. Treatment groups given 7 and 14 mg/kg of Xe-ELIP reduced infarct size. Behavioral outcomes corresponded to changes in infarct volume. Xe-ELIP treatment reduced ischemic neuronal cell death via activation of both MAPK and Akt. Elevated BDNF expression was shown following Xe-ELIP delivery. CONCLUSION: This study demonstrates the therapeutic efficacy of Xe-ELIP administered within 5 h after stroke onset with an optimal dosage range of 7-14 mg/kg for maximal neuroprotection.

In vivo therapeutic gas delivery for neuroprotection with echogenic liposomes.

BACKGROUND: Ischemia-related neurological injury is a primary cause of stroke disability. Studies have demonstrated that xenon (Xe) may have potential as an effective and nontoxic neuroprotectant. Xe delivery is, however, hampered by lack of suitable administration methods. We have developed a pressurization-freeze method to encapsulate Xe into echogenic liposomes (Xe-ELIP) and have modulated local gas release with transvascular ultrasound exposure. METHODS AND RESULTS: Fifteen microliters of Xe were encapsulated into each 1 mg of liposomes (70% Xe and 30% argon). Xe delivery from Xe-ELIP into cells and consequent neuroprotective effects were evaluated with oxygen/glucose-deprived and control neuronal cells in vitro. Xe-ELIP were administered into Sprague-Dawley rats intravenously or intra-arterially after right middle cerebral artery occlusion. One-megahertz low-amplitude (0.18 MPa) continuous wave ultrasound directed onto the internal carotid artery triggered Xe release from circulating Xe-ELIP. Effects of Xe delivery on ischemia-induced neurological injury and disability were evaluated. Xe-ELIP delivery to oxygen/glucose-deprived neuronal cells improved cell viability in vitro and resulted in a 48% infarct volume decrease in vivo. Intravenous Xe-ELIP administration in combination with the ultrasound directed onto the carotid artery enhanced local Xe release from circulating Xe-ELIP and demonstrated 75% infarct volume reduction. This was comparable to the effect after intra-arterial administration. Behavioral tests on limb placement and grid and beam walking correlated with infarct reduction. CONCLUSIONS: This novel methodology may provide a noninvasive strategy for ultrasound-enhanced local therapeutic gas delivery for cerebral ischemia-related injury while minimizing systemic side effects.