Browsing by Author "Floyd, Jason L."

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    Bone Marrow-Derived Cells Restore Functional Integrity of the Gut Epithelial and Vascular Barriers in a Model of Diabetes and ACE2 Deficiency
    (AHA, 2019-11-08) Duan, Yaqian; Prasad, Ram; Feng, Dongni; Beli, Eleni; Calzi, Sergio Li; Longhini, Ana Leda F.; Lamendella, Regina; Floyd, Jason L.; Dupont, Mariana; Noothi, Sunil K.; Sreejit, Gopal Krishan; Athmanathan, Baskaran; Wright, Justin; Jensen, Amanda R.; Oudit, Gavin Y.; Markel, Troy A.; Nagareddy, Prabhakara R; Obukhov, Alexander G.; Grant, Maria B.; Anatomy and Cell Biology, School of Medicine
    Rationale: There is incomplete knowledge of the impact of bone marrow (BM) cells on the gut microbiome and gut barrier function. Objective: We postulated that diabetes and systemic angiotensin-converting enzyme 2 (ACE2) deficiency would synergize to adversely impact both the microbiome and gut barrier function. Methods and Results: Bacterial 16S rRNA sequencing and metatranscriptomic analysis were performed on fecal samples from WT, ACE2−/y, Akita (type 1 diabetic, T1D), and ACE2−/y-Akita mice. Gut barrier integrity was assessed by immunofluorescence, and BM cell extravasation into the small intestine was evaluated by flow cytometry. In the ACE2−/y-Akita or Akita mice, the disrupted barrier was associated with reduced levels of myeloid angiogenic cells (MACs), but no increase in inflammatory monocytes was observed within the gut parenchyma. Genomic and metatranscriptomic analysis of the microbiome of ACE2−/y-Akita mice demonstrated a marked increase in peptidoglycan (PGN) producing bacteria. When compared to control cohorts treated with saline, intraperitoneal administration of MACs significantly decreased the microbiome gene expression associated with PGN biosynthesis and restored epithelial and endothelial gut barrier integrity. Also indicative of diabetic gut barrier dysfunction, increased levels of PGN and intestinal fatty acid binding protein-2 (FABP-2) were observed in plasma of human subjects with T1D (n=21) and Type 2 diabetes (T2D, n=23) compared to non-diabetic controls (n=23). Using human retinal endothelial cells, we determined that PGN activates a non-canonical Toll-like receptor-2 (TLR2) associated MyD88-ARNO-ARF6 signaling cascade, resulting in destabilization of p120-catenin and internalization of VE-cadherin as a mechanism of deleterious impact of PGN on the endothelium. Conclusion: We demonstrate for the first time that the defect in gut barrier function and dysbiosis in ACE2−/y-Akita mice can be favorably impacted by exogenous administration of MACs.
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    Microbial Signatures in The Rodent Eyes With Retinal Dysfunction and Diabetic Retinopathy
    (Association for Research in Vision and Ophthalmology, 2022) Prasad, Ram; Asare-Bediko, Bright; Harbour, Angela; Floyd, Jason L.; Chakraborty, Dibyendu; Duan, Yaqian; Lamendella, Regina; Wright, Justin; Grant, Maria B.; Anatomy, Cell Biology and Physiology, School of Medicine
    Purpose: The gut microbiome has been linked to disease pathogenesis through their interaction in metabolic, endocrine, and immune functions. The goal of this study was to determine whether the gut and plasma microbiota could transfer microbes to the retina in type 1 diabetic mice with retinopathy. Methods: We analyzed the fecal, plasma, whole globe, and retina microbiome in Akita mice and compared with age-matched wild-type (WT) mice using 16S rRNA sequencing and metatranscriptomic analysis. To eliminate the contribution of the ocular surface and plasma microbiome, mice were perfused with sterile saline solution, the whole globes were extracted, and the neural retina was removed under sterile conditions for retinal microbiome. Results: Our microbiome analysis revealed that Akita mice demonstrated a distinct pattern of microbes within each source: feces, plasma, whole globes, and retina. WT mice and Akita mice experienced transient bacteremia in the plasma and retina. Bacteria were identified in the retina of the Akita mice, specifically Corynebacterium, Pseudomonas, Lactobacillus, Staphylococcus, Enterococcus, and Bacillus. Significantly increased levels of peptidoglycan (0.036 ± 0.001 vs. 0.023 ± 0.002; P < 0.002) and TLR2 (3.47 ± 0.15 vs. 1.99 ± 0.07; P < 0.0001) were observed in the retina of Akita mice compared to WT. Increased IBA+ cells in the retina, reduced a- and b-waves on electroretinography, and increased acellular capillary formation demonstrated the presence of retinopathy in the Akita cohort compared to WT mice. Conclusions: Together, our findings suggest that transient bacteremia exists in the plasma and retina of both cohorts. The bacteria found in Akita mice are distinct from WT mice and may contribute to development of retinal inflammation and barrier dysfunction in retinopathy.
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    Specific mesoderm subset derived from human pluripotent stem cells ameliorates microvascular pathology in type 2 diabetic mice
    (American Association for the Advancement of Science, 2022) Gil, Chang-Hyun; Chakraborty, Dibyendu; Vieira, Cristiano P.; Prasain, Nutan; Calzi, Sergio Li; Fortmann, Seth D.; Hu, Ping; Banno, Kimihiko; Jamal, Mohamed; Huang, Chao; Sielski, Micheli S.; Lin, Yang; Huang, Xinxin; Dupont, Mariana D.; Floyd, Jason L.; Prasad, Ram; Longhini, Ana Leda F.; McGill, Trevor J.; Chung, Hyung-Min; Murphy, Michael P.; Kotton, Darrell N.; Boulton, Michael E.; Yoder, Mervin C.; Grant, Maria B.; Pediatrics, School of Medicine
    Human induced pluripotent stem cells (hiPSCs) were differentiated into a specific mesoderm subset characterized by KDR+CD56+APLNR+ (KNA+) expression. KNA+ cells had high clonal proliferative potential and specification into endothelial colony-forming cell (ECFCs) phenotype. KNA+ cells differentiated into perfused blood vessels when implanted subcutaneously into the flank of nonobese diabetic/severe combined immunodeficient mice and when injected into the vitreous of type 2 diabetic mice (db/db mice). Transcriptomic analysis showed that differentiation of hiPSCs derived from diabetics into KNA+ cells was sufficient to change baseline differences in gene expression caused by the diabetic status and reprogram diabetic cells to a pattern similar to KNA+ cells derived from nondiabetic hiPSCs. Proteomic array studies performed on retinas of db/db mice injected with either control or diabetic donor-derived KNA+ cells showed correction of aberrant signaling in db/db retinas toward normal healthy retina. These data provide "proof of principle" that KNA+ cells restore perfusion and correct vascular dysfunction in db/db mice.