Kontrastverstärkter Ultraschall mit VEGFR-2 spezifischem Kontrastmittel zum Monitoring der Therapieeffekte von Regorafenib auf experimentelle kolorektale Adenokarzinome in der Ratte

Zielsetzung In dieser tierexperimentellen Studie wurde das Potential von kontrastverstärktem Ultraschall (CEUS) unter Verwendung des VEGFR-2-spezifischen Kontrastmittels BR55 als Imaging Biomarker zum Monitoring der frühen Therapieeffekte des Tyrosinkinaseinhibitors Regorafenib auf humane Kolonkarzinom-Xenografts in der Ratte untersucht. Die Ergebnisse der CEUS-Untersuchungen wurden dabei jeweils mit etablierten, nicht-invasiven DCE-MRT-Parametern korreliert und immunhistochemisch validiert. Material und Methoden Humane kolorektale Adenokarzinom-Xenografts wurden subkutan in n = 21 (n = 11 Therapiegruppe; n = 10 Kontrollgruppe) weibliche athymische Nacktratten implantiert. Die Tiere erhielten sowohl zur Baseline als auch zum Follow-Up nach einer einwöchigen Therapie mit Regorafenib bzw. einem Placebo CEUS- Untersuchungen mit VEGFR-2-spezifischen Microbubbles sowie direkt anschließende DCE-MRT-Messungen. In den CEUS-Untersuchungen wurde die Tumorperfusion während einer frühen vaskulären Phase (WiAUC) sowie die VEGFR- 2-spezifische Bindung während einer späten, molekularen Phase (SI8min und SI10min) mit einem klinischen Ultraschallgerät und einem 15L8 Linear-Schallkopf ermittelt. In der DCE-MRT wurden die funktionellen Parameter der Tumorperfusion und - vaskularisation (PF und PV) quantifiziert. Zu Validierungszwecken wurden die CEUS- Parameter mit den DCE-MRT-Parametern sowie der Immunhistochemie (CD31, Ki- 67, TUNEL und VEGFR-2-Expression) korreliert. Ergebnisse Der CEUS-Perfusionsparameter WiAUC nahm signifikant (von 116.989 ± 77.048 a.u. auf 30.076 ± 27.095 a.u.; p = 0,005) unter Therapie mit Regorafenib ab, wobei keine signifikanten Veränderungen in der Kontrollgruppe beobachtet wurden (von 133.932 ± 65.960 a.u. auf 84.316 ± 74.144 a.u.; p= 0,093). In der Therapiegruppe war zudem an Tag 7 die Zahl spezifisch gebundener Microbubbles in der späten vaskulären Phase signifikant niedriger als in der Kontrollgruppe (SI8min: 283 ± 191 a.u. vs. 802 ± 460 a.u.; p = 0.006); (SI10min: 226 ± 149 vs. 645 ± 461 a.u.; p= 0,009). PF und PV nahmen in der Therapiegruppe im Therapieverlauf signifikant ab (PF: von 147 ± 58 mL/100mL/min auf 71 ± 15 mL/100mL/min; p = 0,003; PV von 12,5 ± 3,2 % auf 8,6 ± 3,7 % p = 0,037). In der Immunhistochemie zeigte sich eine signifikant niedrigere VEGFR-2-Expression (7,2 ± 1,8 vs. 17,8 ± 4,6; p < 0,001), signifikant weniger CD31- (8,1 ± 3,0 vs. 20,8 ± 5,7, p < 0,001) und Ki-67- positive Zellen (318,7 ± 94,0 vs. 468,0 ± 133,8; p = 0,004) sowie signifikant mehr TUNEL-positive Zellen (672,7 ± 194,0 vs. 357,6 ± 192,0; p = 0,003) in der Therapiegruppe. Die CEUS-Parameter zeigten eine signifikante (p<0,05) Korrelation mit den DCE-MRT- sowie den immunhistochemischen Parametern. Schlussfolgerung Es konnte gezeigt werden, dass CEUS mit BR55 eine multiparametrische, nicht- invasive Charakterisierung der Tumorangiogenese unter Regorafenib-Therapie ermöglicht. Hierdurch könnten zukünftig Therapie Non-Responder schnell und frühzeitig identifiziert werden. Eine effiziente Therapiesteuerung entlastet den Patienten und erlaubt eine zeitgerechte Anpassung des Therapieregimes mit verbesserter Effektivität und Wirtschaftlichkeit. Es bedarf weiterer klinischer Studien, um eine mögliche Implementierung des Verfahrens in den klinischen Alltag zu evaluieren. Die Implementierung validierter Imaging Biomarker zur onkologischen Therapiesteuerung eröffnet einen vielversprechenden Weg für die individualisierte Präzisionsmedizin, die den Patienten konzeptuell in den Mittelpunkt stellt

Contrast-Enhanced Ultrasound with VEGFR2-Targeted Microbubbles for Monitoring Regorafenib Therapy Effects in Experimental Colorectal Adenocarcinomas in Rats with DCE-MRI and Immunohistochemical Validation

Objectives To investigate contrast-enhanced ultrasound (CEUS) with VEGFR2-targeted microbubbles for monitoring therapy effects of regorafenib on experimental colon carcinomas in rats with correlation to dynamic contrast-enhanced MRI (DCE-MRI) and immunohistochemistry. Materials and Methods: Human colorectal adenocarcinoma xenografts (HT-29) were implanted subcutaneously in n =21 (n = 11 therapy group;n = 10 control group) female athymic nude rats (Hsd: RH-Foxn1 (mu)). Animals were imaged at baseline and after a one-week daily treatment with regorafenib or a placebo (10 mg/kg bodyweight), using CEUS with VEGFR2-targeted microbubbles and DCE-MRI. In CEUS tumor perfusion was assessed during an early vascular phase (wash-in area under the curve = WiAUC) and VEGFR2-specific binding during a late molecular phase (signal intensity after 8 (SI8min) and 10 minutes (SI10min)), using a conventional 15L8 linear transducer (transmit frequency 7 MHz, dynamic range 80 dB, depth 25 mm). In DCE-MRI functional parameters plasma flow (PF) and plasma volume (PV) were quantified. For validation purposes, CEUS parameters were correlated with DCE-MRI parameters and immunohistochemical VEGFR2, CD31, Ki-67 and TUNEL stainings. Results: CEUS perfusion parameter WiAUC decreased significantly (116,989 +/- 77,048 a.u. to 30,076 +/- 27,095a.u.;p = 0.005) under therapy with no significant changes (133,932 +/- 65,960 a.u. to 84,316 +/- 74,144 a.u.;p = 0.093) in the control group. In the therapy group, the amount of bound microbubbles in the late phase was significantly lower in the therapy than in the control group on day 7 (SI8min: 283 +/- 191 vs. 802 +/- 460 a.u.;p = 0.006);SI10min: 226 +/- 149 vs. 645 +/- 461 a.u.;p = 0.009). PF and PV decreased significantly (PF: 147 +/- 58 mL/100 mL/min to 71 +/- 15 mL/100 mL/min;p = 0.003;PV: 13 +/- 3% to 9 +/- 4%;p = 0.040) in the therapy group. Immunohistochemistry revealed significantly fewer VEGFR2 (7.2 +/- 1.8 vs. 17.8 +/- 4.6;p < 0.001), CD31 (8.1 +/- 3.0 vs. 20.8 +/- 5.7;p < 0.001) and Ki-67 (318.7 +/- 94.0 vs. 468.0 +/- 133.8;p = 0.004) and significantly more TUNEL (672.7 +/- 194.0 vs. 357.6 +/- 192.0;p = 0.003) positive cells in the therapy group. CEUS parameters showed significant (p < 0.05) correlations to DCE-MRI parameters and immunohistochemistry. Conclusions CEUS with VEGFR2-targeted microbubbles allowed for monitoring regorafenib functional and molecular therapy effects on experimental colorectal adenocarcinomas with a significant decline of CEUS and DCE-MRI perfusion parameters as well as a significant reduction of specifically bound microbubbles under therapy, consistent with a reduced expression of VEGFR2

Genome-wide association analyses identify new Brugada syndrome risk loci and highlight a new mechanism of sodium channel regulation in disease susceptibility.

Brugada syndrome (BrS) is a cardiac arrhythmia disorder associated with sudden death in young adults. With the exception of SCN5A, encoding the cardiac sodium channel Na1.5, susceptibility genes remain largely unknown. Here we performed a genome-wide association meta-analysis comprising 2,820 unrelated cases with BrS and 10,001 controls, and identified 21 association signals at 12 loci (10 new). Single nucleotide polymorphism (SNP)-heritability estimates indicate a strong polygenic influence. Polygenic risk score analyses based on the 21 susceptibility variants demonstrate varying cumulative contribution of common risk alleles among different patient subgroups, as well as genetic associations with cardiac electrical traits and disorders in the general population. The predominance of cardiac transcription factor loci indicates that transcriptional regulation is a key feature of BrS pathogenesis. Furthermore, functional studies conducted on MAPRE2, encoding the microtubule plus-end binding protein EB2, point to microtubule-related trafficking effects on Na1.5 expression as a new underlying molecular mechanism. Taken together, these findings broaden our understanding of the genetic architecture of BrS and provide new insights into its molecular underpinnings

Alteration in corneal topography asymmetry indices following corneal refractive surgery

Purpose We aimed to follow up corneal refractive asymmetry index changes following refractive surgery for cornea after correction of low-grade myopia and to determine which technique causes marked affection of the indices. Patients and methods This prospective study conducted from October 2020 to December 2021 included two groups of patients, the photorefractive keratectomy (PRK) group and the laser in-situ keratomileusis (LASIK) group. Every patient in each group had to follow a schedule of visits after surgery for the next day, 1 week, 1 month, and after 3 months. Patients who failed to follow this schedule were excluded from the study. Results The PRK group contained 42 eyes with a mean uncorrected visual acuity that improved significantly, corneal power (K-reading) and pupil center thickness reduced significantly. Surface variance index, central keratoconus index, and height decentration index did not change significantly. Vertical asymmetry index and minimum radius of curvature (Rmin) increased significantly while height asymmetry index decreased significantly. Our LASIK group contained 40 eyes that underwent changes similar to those in the PRK group regarding uncorrected visual acuity, the K-reading, and the pupil center thickness. The surface variance index, vertical asymmetry index, and height asymmetry index, height decentration index and Rmin increased significantly while the central keratoconus index decreased significantly. The keratoconus index reduced significantly in both groups. Conclusion The PRK is a more conservative technique than the LASIK technique regarding the affection of corneal asymmetry indices

Histopathology and electron microscopy evaluation of the sildenafil effect on diabetic rats' retinae

Purpose: Although there is increasing evidence that phosphodiesterase-5 (PDE-5) inhibitors modify the effect of diabetes on different tissues, its effect on diabetic retinopathy is not well studied. Methods: Forty male Sprague–Dawley (SD) rats were divided into four groups: group I = control group that received no treatment; group II (diabetic group), in which diabetes was induced by a single streptozotocin injection; group III (sildenafil small dose, SSD), in which diabetes was similarly introduced (however, rats received daily oral 1 mg/kg sildenafil citrate (SC) for 3 months); and group IV (sildenafil large dose, SLD), which was as in group 3, but SC was 2.5 mg/kg. After 3 months, globes were removed and retinae were dissected; one globe from each rat was examined by light microscopy (LM), and the other by electron microscopy (EM). Results: In contrast to the control group, diabetic rats in group II demonstrated well-established diabetic changes in the form of capillary congestion, decreased cell population, hyaline changes of capillary walls, and degenerated nerve fiber layer by LM. Similarly, EM demonstrated photoreceptor degeneration, mitochondrial cristolysis, and vacuolated depleted cells among other features in group II. These diabetic features were less prominent in group III and nearly absent in group IV. Conclusion: SC use in the early stages of DR may prevent/delay diabetic retinopathy development or progression in diabetic rat models, an effect that seems to be dose-related

Chronic sildenafil citrate use decreases retinal vascular endothelial growth factor expression in diabetic rats: a pilot study

Abstract Background Sildenafil citrate (SC) attenuates endothelial dysfunction. However, its effects on diabetic retinopathy (DR), which is mainly a microvascular disease, remain unclear. Vascular endothelial growth factor (VEGF) is known to be a critical mediator of DR. Therefore, we investigated the effects of SC on diabetic retina by measuring VEGF levels. Methods In this study, twenty-eight rats were divided into the following groups: group I, the control group; group II, rats with streptozotocin-induced diabetes; group III, rats with streptozotocin-induced diabetes receiving daily oral sildenafil at 1 mg/kg; and group IV, rats with streptozotocin-induced diabetes receiving high-dose daily sildenafil at 2.5 mg/kg. After 3 months, VEGF was measured in the retina specimen in one eye and the vitreous body in the other eye by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. Results We found that VEGF expression in the retina was low in all rats from groups I and IV and in 30% of rats from group III; 80% of rats in group II demonstrated high VEGF expression in the retinae (P < 0.001). VEGF concentrations in the vitreous body samples were 32 ± 2, 61 ± 4, 44 ± 5, and 36 ± 3 pg/l in groups I–IV, respectively (P < 0.001). Conclusion VEGF decreased significantly in the eyes of diabetic rats after chronic oral sildenafil citrate treatment. SC may have a modifying/attenuating effect on DR. However, further studies are needed to evaluate its use as an adjunctive treatment

Line regression analysis for the correlation between CEUS parameter of VEGFR2-specific binding and immunohistochemical VEGFR2 expression.

<p>Note the significant correlation between the values of SI_8min and the number of VEGFR2 positive stained vessels (Spearman´s ρ = 0.66, p = 0.003) for the therapy and the control group.</p

Individual DCE-MRI values.

<p>Individual DCE-MRI values.</p

Representative CEUS images of a rat with a subcutaneous tumor xenograft (white arrows) under treatment.

<p>Therapy group (red) and control group (blue) at baseline and follow-up. Left side: early vascular phase with BR55 as a functional imaging biomarker; right side: late phase of VEGFR2-specific binding with BR55 as a molecular imaging biomarker 8 minutes after contrast injection. Note the significant lower number of circulating microbubbles in the early vascular phase as well as the significant lower number of bound microbubbles in the late phase at follow-up in the therapy group, compared to baseline and compared to the control group.</p

Individual molecular CEUS values.

<p>Individual molecular CEUS values.</p