BACKGROUNDNovel treatment strategies are needed to improve the structure and function of the myocardium post-infarction. In vitro-matured pluripotent stem cell-derived cardiomyocytes (PSC-CMs) have been shown to be a promising regenerative strategy. We hypothesized that mature PSC-CMs will have anisotropic structure and improved cell alignment when compared to immature PSC-CMs using cardiovascular magnetic resonance (CMR) in a guinea pig model of cardiac injury.METHODSGuinea pigs (n = 16) were cryoinjured on day -10, followed by transplantation of either 10⁸ polydimethylsiloxane (PDMS)-matured PSC-CMs (n = 6) or 10⁸ immature tissue culture plastic (TCP)-generated PSC-CMs (n = 6) on day 0. Vehicle (sham-treated) subjects were injected with a pro-survival cocktail devoid of cells (n = 4), while healthy controls (n = 4) did not undergo cryoinjury or treatment. Animals were sacrificed on either day +14 or day +28 post-transplantation. Animals were imaged ex vivo on a 7T Bruker MRI. A 3D diffusion tensor imaging (DTI) sequence was used to quantify structure via fractional anisotropy (FA), mean diffusivity (MD), and myocyte alignment measured by the standard deviation of the transverse angle (TA).RESULTSMD and FA of mature PDMS grafts demonstrated anisotropy was not significantly different than the healthy control hearts (MD = 1.1 ± 0.12 × 10^-3 mm²/s vs 0.93 ± 0.01 × 10^-3 mm²/s, p = 0.4 and FA = 0.22 ± 0.05 vs 0.26 ± 0.001, p = 0.5). Immature TCP grafts exhibited significantly higher MD than the healthy control (1.3 ± 0.08 × 10^-3 mm²/s, p < 0.05) and significantly lower FA than the control (0.12 ± 0.02, p < 0.05) but were not different from mature PDMS grafts in this small cohort. TA of healthy controls showed low variability and was not significantly different than mature PDMS grafts (p = 0.4) while immature TCP grafts were significantly different (p < 0.001). DTI parameters of mature graft tissue trended toward that of the healthy myocardium, indicating the grafted cardiomyocytes may have a similar phenotype to healthy tissue. Contrast-enhanced magnetic resonance images corresponded well to histological staining, demonstrating a non-invasive method of localizing the repopulated cardiomyocytes within the scar.CONCLUSIONSThe DTI measures within graft tissue were indicative of anisotropic structure and showed greater myocyte organization compared to the scarred territory. These findings show that MRI is a valuable tool to assess the structural impacts of regenerative therapies.

2023-11-11·The Journal of Infectious Diseases

Cost-effective In Vivo and In Vitro Mouse Models for Evaluating Anticryptosporidial Drug Efficacy: Assessing Vorinostat, Docetaxel, and Baicalein

Article

作者: Liu, Mingxiao ; Yin, Jigang ; Zhang, Ying ; Zhang, Di ; Wu, Xiaodong ; Wang, Dongqiang ; Zhu, Guan

AbstractBackgroundCryptosporidiosis is a significant diarrheal disease in humans and animals. Immunodeficient mice are the primary small animal models, but their high costs and specialized breeding/housing requirements limit in vivo drug testing. Numerous anticryptosporidial lead compounds identified in vitro remain untested in vivo.MethodsCryptosporidium tyzzeri, a natural mouse parasite closely related to Cryptosporidium parvum and Cryptosporidium hominis, was isolated to establish an infection model in immunocompetent mice. The model was validated using classic anticryptosporidial drugs (paromomycin and nitazoxanide) and then employed to assess the efficacy of 3 new leads (vorinostat, docetaxel, and baicalein). An in vitro culture of C. tyzzeri was also developed to complement the animal model.ResultsChronic C. tyzzeri infection was established in chemically immunosuppressed wild-type mice. Paromomycin (1000 mg/kg/d) and nitazoxanide (100 mg/kg/d) demonstrated efficacy against C. tyzzeri. Vorinostat (30 mg/kg/d), docetaxel (25 mg/kg/d), and baicalein (50 mg/kg/d) were highly effective against C. tyzzeri infection. In vitro, nitazoxanide, vorinostat, docetaxel, and baicalein exhibited low to submicromolar efficacy against C. tyzzeri.ConclusionsNovel in vivo and in vitro models have been developed for cost-effective anticryptosporidial drug testing. Vorinostat, docetaxel, and baicalein show potential for repurposing and/or optimization for developing new anticryptosporidial drugs.

2023-11-01·British journal of pharmacology

Pharmacological inhibition of TRPV2 attenuates phagocytosis and lipopolysaccharide-induced migration of primary macrophages.

Article

作者: Deckers, Anke ; Gräßle, Simone ; Paulig, Andrea ; Bräse, Stefan ; Jung, Nicole ; Raudszus, Rick ; Urban, Nicole ; Vanderheiden, Sylvia ; Hill, Kerstin ; Schaefer, Michael

BACKGROUND AND PURPOSEIn macrophages, transient receptor potential vanilloid 2 (TRPV2) channel contributes to various cellular processes such as cytokine production, differentiation, phagocytosis and migration. Due to a lack of selective pharmacological tools, its function in immunological processes is not well understood and the identification of novel and selective TRPV2 modulators is highly desirable.EXPERIMENTAL APPROACHNovel and selective TRPV2 modulators were identified by screening a compound library using Ca²⁺ influx assays with human embryonic kidney 293 (HEK293) cells heterologously expressing rat TRPV2. Hits were further characterized and validated with Ca²⁺ influx and electrophysiological assays. Phagocytosis and migration of macrophages were analysed and the contribution of TRPV2 to the generation of Ca²⁺ microdomains was studied by total internal reflection fluorescence microscopy (TIRFM).KEY RESULTSThe compound IV2-1, a dithiolane derivative (1,3-dithiolan-2-ylidene)-4-methyl-5-phenylpentan-2-one), is a potent inhibitor of heterologously expressed TRPV2 channels (IC₅₀ = 6.3 ± 0.7 μM) but does not modify TRPV1, TRPV3 or TRPV4 channels. IV2-1 also inhibits TRPV2-mediated Ca²⁺ influx in macrophages. IV2-1 inhibits macrophage phagocytosis along with valdecoxib and after siRNA-mediated knockdown. Moreover, TRPV2 inhibition inhibits lipopolysaccharide-induced migration of macrophages whereas TRPV2 activation promotes migration. After activation, TRPV2 shapes Ca²⁺ microdomains predominantly at the margin of macrophages, which are important cellular regions to promote phagocytosis and migration.CONCLUSIONS AND IMPLICATIONSIV2-1 is a novel TRPV2-selective blocker and underline the role of TRPV2 in macrophage-mediated phagocytosis and migration. Furthermore, we provide evidence that TRPV2 activation generates Ca²⁺ microdomains, which may be involved in phagocytosis and migration of macrophages.

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