Microvascular flow imaging of fibroids: A prospective pilot study.

INTRODUCTION: Imaging fibroid vascularity may predict fibroid growth and aid to determine most appropriate therapy. Microvascular (MV) flow imaging is relatively new and is able to detect slow flow in small vessels. Data on feasibility, reproducibility, and reliability of MV-flow imaging in fibroids is lacking. The purpose of our study was to determine the reproducibility of MV-flow imaging and to explore this technique for clinical practice for assessing blood flow in fibroids. MATERIAL AND METHODS: Thirty patients with one or multiple fibroids (diameter 1.5-12.0 cm) were prospectively included. Transvaginal ultrasound scanning was performed in B-mode, 2D MV-Flow™, 2D and 3D power Doppler mode (HERA W10, Samsung) by two experienced gynecologists at a tertiary care clinic from February to December 2021. The primary outcome was intra- and interobserver agreement of the vascular index (VI) and color score (CS). The following parameters: '2D MV-flow VI', '3DPD VI', '2D MV-flow CS' and '2DPD CS' were measured offline in the center, pseudocapsule, and entire fibroid. Secondary offline outcomes for exploring 2D MV-flow for clinical practice, included (1) ability to discern vascular structures, (2) assessing the degree of vascularity via CS and calculating a VI, and (3) determining penetration depth of the ultrasound signal in both power Doppler and MV-flow imaging. RESULTS: All scans of the 30 included patients were of sufficient quality to analyze. Inter- and intra-observer correlations of all studied parameters were good to excellent, both for 2D MV-flow and 2D power Doppler (intercorrelation coefficient 0.992-0.996). Using 2D MV-flow different vascular structures were visible in detail, in contrary to using 2D and 3D power Doppler. In significantly more fibroids central flow could be visualized using 2D MV-flow (63%) than with 2D power Doppler (13%, p = 0.001). Finally, penetration of the ultrasound signal was deeper using 2D MV-flow (3.92 cm) than with 2D power Doppler (2.95 cm, p = 0.001). CONCLUSIONS: Using 2D MV-flow imaging for determining vascularity is highly reproducible. It has potential added value for clinical practice as it depicts detailed vascular structures and the degree of vascularity, especially in the center of the fibroid.

Increased Angiogenesis and Lymphangiogenesis in Adenomyosis Visualized by Multiplex Immunohistochemistry.

There is evidence for increased angiogenesis in the (ectopic) endometrium of adenomyosis patients under the influence of vascular endothelial growth factor (VEGF). VEGF stimulates both angiogenesis and lymph-angiogenesis. However, information on lymph vessels in the (ectopic) endometrium of adenomyosis patients is lacking. In this retrospective matched case-control study, multiplex immunohistochemistry was performed on thirty-eight paraffin embedded specimens from premenopausal women who had undergone a hysterectomy at the Amsterdam UMC between 2001 and 2018 to investigate the evidence for (lymph) angiogenesis in the (ectopic) endometrium or myometrium of patients with adenomyosis versus controls with unrelated pathologies. Baseline characteristics of both groups were comparable. In the proliferative phase, the blood and lymph vessel densities were, respectively, higher in the ectopic and eutopic endometrium of patients with adenomyosis than in the endometrium of controls. The relative number of blood vessels without α-smooth muscle actinin (α SMA) was higher in the eutopic and ectopic endometrium of adenomyosis patients versus controls. The level of VEGF staining intensity was highest in the myometrium but did not differ between patients with adenomyosis or controls. The results indicate increased angiogenesis and lymphangiogenesis in the (ectopic) endometrium affected by adenomyosis. The clinical relevance of our findings should be confirmed in prospective clinical studies.

StemBell therapy stabilizes atherosclerotic plaques after myocardial infarction.

BACKGROUND AIMS: After a myocardial infarction (MI) atherosclerosis is accelerated leading to destabilization of the atherosclerotic plaque. mesenchymal stromal cells are a promising therapeutic option for atherosclerosis. Previously, we demonstrated a novel stem cell delivery technique, with adipose stem cells coupled to microbubbles (i.e., StemBells) as therapy after MI. In this study, we aim to investigate the effect of StemBell therapy on atherosclerotic plaques in an atherosclerotic mouse model after MI. METHODS: MI was induced in atherosclerotic Apolipoprotein E-deficient mice that were fed a high-fat Western diet. Six days post-MI, the mice received either 5 × 105/100 µL StemBells or vehicle intravenously. The effects of StemBell treatment on the size and stability of aortic root atherosclerotic plaques and the infarcted heart were determined 28 days post-MI via (immuno)histological analyses. Moreover, monocyte subtypes and lipids in the blood were studied. RESULTS: StemBell treatment resulted in significantly increased cap thickness, decreased intra-plaque macrophage density and increased percentage of intra-plaque anti-inflammatory macrophages and chemokines, without affecting plaque size and serum cholesterol/triglycerides. Furthermore, StemBell treatment significantly increased the percentage of anti-inflammatory macrophages within the infarcted myocardium but did not affect cardiac function nor infarct size. Finally, also the average percentage of anti-inflammatory monocytes in the circulation was increased after StemBell therapy. DISCUSSION: StemBell therapy increased cap thickness and decreased intra-plaque inflammation after MI, indicative of stabilized atherosclerotic plaque. It also induced a shift of circulating monocytes and intra-plaque and intra-cardiac macrophages towards anti-inflammatory phenotypes. Hence, StemBell therapy may be a therapeutic option to prevent atherosclerosis acceleration after MI.

Use of Contrast-Enhanced Ultrasound in the Assessment of Uterine Fibroids: A Feasibility Study.

Contrast-enhanced ultrasound (CEUS) is an innovative ultrasound technique capable of visualizing both the macro- and microvasculature of tissues. In this prospective pilot study, we evaluated the feasibility of using CEUS to visualize the microvasculature of uterine fibroids and compared CEUS with conventional ultrasound. Four women with fibroids underwent gray-scale ultrasound, sonoelastography and power/color Doppler scans followed by CEUS examination. Analysis of CEUS images revealed initial perfusion of the peripheral rim, that is, a pseudo-capsule, followed by enhancement of the entire lesion through vessels traveling from the exterior to the interior of the fibroid. The pseudo-capsules exhibited slight hyper-enhancement, making a clear delineation of the fibroids possible. The centers of three fibroids exhibited areas lacking vascularization, information not obtainable with the other imaging techniques. CEUS is a feasible technique for imaging and quantifying the microvasculature of fibroids. In comparison with conventional ultrasound imaging modalities, CEUS can provide additional diagnostic information based on the microvasculature.

Homocysteine-induced apoptosis in endothelial cells coincides with nuclear NOX2 and peri-nuclear NOX4 activity.

Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01-2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47(phox) expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47(phox), and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47(phox) in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47(phox) was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.

Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation.

Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4- and 70-kDa dextrans through the cytosol, and localization of 155- and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4- to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size.