JNK2 in myeloid cells impairs insulin's vasodilator effects in muscle during early obesity development through perivascular adipose tissue dysfunction.

Reduced vasodilator properties of insulin in obesity are caused by changes in perivascular adipose tissue and contribute to microvascular dysfunction in skeletal muscle. The causes of this dysfunction are unknown. The effects of a short-term Western diet on JNK2-expressing cells in perivascular adipose tissue (PVAT) on insulin-induced vasodilation and perfusion of skeletal muscle were assessed. In vivo, 2 wk of Western diet (WD) reduced whole body insulin sensitivity and insulin-stimulated muscle perfusion, determined using contrast ultrasonography during the hyperinsulinemic clamp. Ex vivo, WD triggered accumulation of PVAT in skeletal muscle and blunted its ability to facilitate insulin-induced vasodilation. Labeling of myeloid cells with green fluorescent protein identified bone marrow as a source of PVAT in muscle. To study whether JNK2-expressing inflammatory cells from bone marrow were involved, we transplanted JNK2-/- bone marrow to WT mice. Deletion of JNK2 in bone marrow rescued the vasodilator phenotype of PVAT during WD exposure. JNK2 deletion in myeloid cells prevented the WD-induced increase in F4/80 expression. Even though WD and JNK2 deletion resulted in specific changes in gene expression of PVAT; epididymal and subcutaneous adipose tissue; expression of tumor necrosis factor-α, interleukin-1ß, interleukin-6, or protein inhibitor of STAT1 was not affected. In conclusion, short-term Western diet triggers infiltration of JNK2-positive myeloid cells into PVAT, resulting in PVAT dysfunction, nonclassical inflammation, and loss of insulin-induced vasodilatation in vivo and ex vivo.NEW & NOTEWORTHY We demonstrate that in the earliest phase of weight gain, changes in perivascular adipose tissue in muscle impair insulin-stimulated muscle perfusion. The hallmark of these changes is infiltration by inflammatory cells. Deletion of JNK2 from the bone marrow restores the function of perivascular adipose tissue to enhance insulin's vasodilator effects in muscle, showing that the bone marrow contributes to regulation of muscle perfusion.

Viability of endothelial cells after ultrasound-mediated sonoporation: Influence of targeting, oscillation, and displacement of microbubbles.

Microbubbles (MBs) have been shown to create transient or lethal pores in cell membranes under the influence of ultrasound, known as ultrasound-mediated sonoporation. Several studies have reported enhanced drug delivery or local cell death induced by MBs that are either targeted to a specific biomarker (targeted microbubbles, tMBs) or that are not targeted (non-targeted microbubbles, ntMBs). However, both the exact mechanism and the optimal acoustic settings for sonoporation are still unknown. In this study we used real-time uptake patterns of propidium iodide, a fluorescent cell impermeable model drug, as a measure for sonoporation. Combined with high-speed optical recordings of MB displacement and ultra-high-speed recordings of MB oscillation, we aimed to identify differences in MB behavior responsible for either viable sonoporation or cell death. We compared ntMBs and tMBs with identical shell compositions exposed to long acoustic pulses (500-50,000cycles) at various pressures (150-500kPa). Propidium iodide uptake highly correlated with cell viability; when the fluorescence intensity still increased 120s after opening of the pore, this resulted in cell death. Higher acoustic pressures and longer cycles resulted in more displacing MBs and enhanced sonoporation. Non-displacing MBs were found to be the main contributor to cell death, while displacement of tMBs enhanced reversible sonoporation and preserved cell viability. Consequently, each therapeutic application requires different settings: non-displacing ntMBs or tMBs are advantageous for therapies requiring cell death, especially at 500kPa and 50,000cycles, whereas short acoustic pulses causing limited displacement should be used for drug delivery.

Globular adiponectin controls insulin-mediated vasoreactivity in muscle through AMPKα2.

Decreased tissue perfusion increases the risk of developing insulin resistance and cardiovascular disease in obesity, and decreased levels of globular adiponectin (gAdn) have been proposed to contribute to this risk. We hypothesized that gAdn controls insulin's vasoactive effects through AMP-activated protein kinase (AMPK), specifically its α2 subunit, and studied the mechanisms involved. In healthy volunteers, we found that decreased plasma gAdn levels in obese subjects associate with insulin resistance and reduced capillary perfusion during hyperinsulinemia. In cultured human microvascular endothelial cells (HMEC), gAdn increased AMPK activity. In isolated muscle resistance arteries gAdn uncovered insulin-induced vasodilation by selectively inhibiting insulin-induced activation of ERK1/2, and the AMPK inhibitor compound C as well as genetic deletion of AMPKα2 blunted insulin-induced vasodilation. In HMEC deletion of AMPKα2 abolished insulin-induced Ser(1177) phosphorylation of eNOS. In mice we confirmed that AMPKα2 deficiency decreases insulin sensitivity, and this was accompanied by decreased muscle microvascular blood volume during hyperinsulinemia in vivo. This impairment was accompanied by a decrease in arterial Ser(1177) phosphorylation of eNOS, which closely related to AMPK activity. In conclusion, globular adiponectin controls muscle perfusion during hyperinsulinemia through AMPKα2, which determines the balance between NO and ET-1 activity in muscle resistance arteries. Our findings provide a novel mechanism linking reduced gAdn-AMPK signaling to insulin resistance and impaired organ perfusion.

Ultrafast dynamics of the acoustic vaporization of phase-change microdroplets.

Acoustically sensitive emulsions are a promising tool for medical applications such as localized drug delivery. The physical mechanisms underlying the ultrasound-triggered nucleation and subsequent vaporization of these phase-change emulsions are largely unexplored. Here, the acoustic vaporization of individual micron-sized perfluoropentane (PFP) droplets is studied at a nanoseconds timescale. Highly diluted emulsions of PFP-in-water and oil-in-PFP-in-water droplets, ranging from 3.5 to 11 µm in radius, were prepared and the nucleation and growth of the vapor bubbles was imaged at frame rates of up to 20 Mfps. The droplet vaporization dynamics was observed to have three distinct regimes: (1) prior to nucleation, a regime of droplet deformation and oscillatory translations within the surrounding fluid along the propagation direction of the applied ultrasound; (2) a regime characterized by the rapid growth of a vapor bubble enhanced by ultrasound-driven rectified heat transfer; and (3) a final phase characterized by a relatively slow expansion, after ultrasound stops, that is fully dominated by heat transfer. A method to measure the moment of inception of the nucleation event with respect to the phase of the ultrasound wave is proposed. A simple physical model captures quantitatively all of the features of the subsequent vapor bubble growth.

Secondary bjerknes forces deform targeted microbubbles.

In this study, we investigated the effect of secondary Bjerknes forces on targeted microbubbles using high-speed optical imaging. We observed that targeted microbubbles attached to an underlying surface and subject to secondary Bjerknes forces deform in the direction of their neighboring bubble, thereby tending toward a prolate shape. The deformation induces an elastic restoring force, causing the bubbles to recoil back to their equilibrium position; typically within 100 µs after low-intensity ultrasound application. The temporal dynamics of the recoil was modeled as a simple mass-spring system, from which a value for the effective spring constant k of the order 10(-3) Nm(-1) was obtained. Moreover, the translational dynamics of interacting targeted microbubbles was predicted by a hydrodynamic point particle model, including a value of the spring stiffness k of the very same order as derived experimentally from the recoiling curves. For higher acoustic pressures, secondary Bjerknes forces rupture the molecular adhesion of the bubbles to the surface. We used this mutual attraction to quantify the binding force between a single biotinylated microbubble and an avidin-coated surface, which was found to be between 0.9 and 2 nanonewtons (nN). The observation of patches of lipids left at the initial binding site suggests that lipid anchors are pulled out of the microbubble shell, rather than biotin molecules unbinding from avidin. Understanding the effect of ultrasound application on targeted microbubbles is crucial for further advances in the realm of molecular imaging.

Brandaris 128 ultra-high-speed imaging facility: 10 years of operation, updates, and enhanced features.

The Brandaris 128 ultra-high-speed imaging facility has been updated over the last 10 years through modifications made to the camera's hardware and software. At its introduction the camera was able to record 6 sequences of 128 images (500 × 292 pixels) at a maximum frame rate of 25 Mfps. The segmented mode of the camera was revised to allow for subdivision of the 128 image sensors into arbitrary segments (1-128) with an inter-segment time of 17 µs. Furthermore, a region of interest can be selected to increase the number of recordings within a single run of the camera from 6 up to 125. By extending the imaging system with a laser-induced fluorescence setup, time-resolved ultra-high-speed fluorescence imaging of microscopic objects has been enabled. Minor updates to the system are also reported here.