Browsing by Author "Mohanty, Sujit K."

Now showing 1 - 7 of 7
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    Cutaneous Epithelial to Mesenchymal Transition Activator ZEB1 Regulates Wound Angiogenesis and Closure in a Glycemic Status–Dependent Manner
    (American Diabetes Association, 2019-11) Singh, Kanhaiya; Sinha, Mithun; Pal, Durba; Tabasum, Saba; Gnyawali, Surya C.; Khona, Dolly; Sarkar, Subendu; Mohanty, Sujit K.; Soto-Gonzalez, Fidel; Khanna, Savita; Roy, Sashwati; Sen, Chandan K.; Surgery, School of Medicine
    Epithelial to mesenchymal transition (EMT) and wound vascularization are two critical interrelated processes that enable cutaneous wound healing. Zinc finger E-box binding homeobox 1 (ZEB1), primarily studied in the context of tumor biology, is a potent EMT activator. ZEB1 is also known to contribute to endothelial cell survival as well as stimulate tumor angiogenesis. The role of ZEB1 in cutaneous wounds was assessed using Zeb1+/− mice, as Zeb1−/− mice are not viable. Quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomics was used to elucidate the effect of elevated ZEB1, as noted during hyperglycemia. Under different glycemic conditions, ZEB1 binding to E-cadherin promoter was investigated using chromatin immunoprecipitation. Cutaneous wounding resulted in loss of epithelial marker E-cadherin with concomitant gain of ZEB1. The dominant proteins downregulated after ZEB1 overexpression functionally represented adherens junction pathway. Zeb1+/− mice exhibited compromised wound closure complicated by defective EMT and poor wound angiogenesis. Under hyperglycemic conditions, ZEB1 lost its ability to bind E-cadherin promoter. Keratinocyte E-cadherin, thus upregulated, resisted EMT required for wound healing. Diabetic wound healing was improved in ZEB+/− as well as in db/db mice subjected to ZEB1 knockdown. This work recognizes ZEB1 as a key regulator of cutaneous wound healing that is of particular relevance to diabetic wound complication.
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    Endothelial Phospholipase Cγ2 Improves Outcomes of Diabetic Ischemic Limb Rescue Following VEGF Therapy
    (American Diabetes Association, 2022) Rustagi, Yashika; Abouhashem, Ahmed S.; Verma, Priyanka; Verma, Sumit S.; Hernandez, Edward; Liu, Sheng; Kumar, Manishekhar; Guda, Poornachander R.; Srivastava, Rajneesh; Mohanty, Sujit K.; Kacar, Sedat; Mahajan, Sanskruti; Wanczyk, Kristen E.; Khanna, Savita; Murphy, Michael P.; Gordillo, Gayle M.; Roy, Sashwati; Wan, Jun; Sen, Chandan K.; Singh, Kanhaiya; Medicine, School of Medicine
    Therapeutic vascular endothelial growth factor (VEGF) replenishment has met with limited success for the management of critical limb-threatening ischemia. To improve outcomes of VEGF therapy, we applied single-cell RNA sequencing (scRNA-seq) technology to study the endothelial cells of the human diabetic skin. Single-cell suspensions were generated from the human skin followed by cDNA preparation using the Chromium Next GEM Single-cell 3' Kit v3.1. Using appropriate quality control measures, 36,487 cells were chosen for downstream analysis. scRNA-seq studies identified that although VEGF signaling was not significantly altered in diabetic versus nondiabetic skin, phospholipase Cγ2 (PLCγ2) was downregulated. The significance of PLCγ2 in VEGF-mediated increase in endothelial cell metabolism and function was assessed in cultured human microvascular endothelial cells. In these cells, VEGF enhanced mitochondrial function, as indicated by elevation in oxygen consumption rate and extracellular acidification rate. The VEGF-dependent increase in cell metabolism was blunted in response to PLCγ2 inhibition. Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCγ2 in type 1 (streptozotocin-injected) and type 2 (db/db) diabetic ischemic tissue. Nonviral topical tissue nanotransfection technology (TNT) delivery of CDH5 promoter-driven PLCγ2 open reading frame promoted the rescue of hindlimb ischemia in diabetic mice. Improvement of blood flow was also associated with higher abundance of VWF+/CD31+ and VWF+/SMA+ immunohistochemical staining. TNT-based gene delivery was not associated with tissue edema, a commonly noted complication associated with proangiogenic gene therapies. Taken together, our study demonstrates that TNT-mediated delivery of endothelial PLCγ2, as part of combination gene therapy, is effective in diabetic ischemic limb rescue.
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    Epigenetic basis of diabetic vasculopathy
    (Frontiers Media, 2022-12-09) Bhamidipati, Theja; Kumar, Manishekhar; Verma, Sumit S.; Mohanty, Sujit K.; Kacar, Sedat; Reese, Diamond; Martinez, Michelle M.; Kamocka, Malgorzata M.; Dunn, Kenneth W.; Sen, Chandan K.; Singh, Kanhaiya; Surgery, School of Medicine
    Type 2 diabetes mellitus (T2DM) causes peripheral vascular disease because of which several blood-borne factors, including vital nutrients fail to reach the affected tissue. Tissue epigenome is sensitive to chronic hyperglycemia and is known to cause pathogenesis of micro- and macrovascular complications. These vascular complications of T2DM may perpetuate the onset of organ dysfunction. The burden of diabetes is primarily because of a wide range of complications of which nonhealing diabetic ulcers represent a major component. Thus, it is imperative that current research help recognize more effective methods for the diagnosis and management of early vascular injuries. This review addresses the significance of epigenetic processes such as DNA methylation and histone modifications in the evolution of macrovascular and microvascular complications of T2DM.
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    Genome-wide DNA hypermethylation opposes healing in patients with chronic wounds by impairing epithelial-mesenchymal transition
    (The American Society for Clinical Investigation, 2022) Singh, Kanhaiya; Rustagi, Yashika; Abouhashem, Ahmed S.; Tabasum, Saba; Verma, Priyanka; Hernandez, Edward; Pal, Durba; Khona, Dolly K.; Mohanty, Sujit K.; Kumar, Manishekhar; Srivastava, Rajneesh; Guda, Poornachander R.; Verma, Sumit S.; Mahajan, Sanskruti; Killian, Jackson A.; Walker, Logan A.; Ghatak, Subhadip; Mathew-Steiner, Shomita S.; Wanczyk, Kristen E.; Liu, Sheng; Wan, Jun; Yan, Pearlly; Bundschuh, Ralf; Khanna, Savita; Gordillo, Gayle M.; Murphy, Michael P.; Roy, Sashwati; Sen, Chandan K.; Surgery, School of Medicine
    An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. An unbiased whole-genome methylome was studied in the wound-edge tissue of patients with chronic wounds. A total of 4,689 differentially methylated regions (DMRs) were identified in chronic wound-edge skin compared with unwounded human skin. Hypermethylation was more frequently observed (3,661 DMRs) in the chronic wound-edge tissue compared with hypomethylation (1,028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial-mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA-Seq analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified 4 genes, ADAM17, NOTCH, TWIST1, and SMURF1, that functionally represent the EMT pathway. Single-cell RNA-Seq studies revealed that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces wound-edge gene methylation and that 5'-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the wound edge was achieved by a tissue nanotransfection-based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene silencing represents a productive therapeutic strategy to improve wound closure.
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    Human fetal dermal fibroblast-myeloid cell diversity is characterized by dominance of pro-healing Annexin1-FPR1 signaling
    (Elsevier, 2023-08-02) Srivastava, Rajneesh; Singh, Kanhaiya; Abouhashem, Ahmed S.; Kumar, Manishekhar; Kacar, Sedat; Verma, Sumit S.; Mohanty, Sujit K.; Sinha, Mithun; Ghatak, Subhadip; Xuan, Yi; Sen, Chandan K.; Surgery, School of Medicine
    Fetal skin achieves scarless wound repair. Dermal fibroblasts play a central role in extracellular matrix deposition and scarring outcomes. Both fetal and gingival wound repair share minimal scarring outcomes. We tested the hypothesis that compared to adult skin fibroblasts, human fetal skin fibroblast diversity is unique and partly overlaps with gingival skin fibroblasts. Human fetal skin (FS, n = 3), gingiva (HGG, n = 13), and mature skin (MS, n = 13) were compared at single-cell resolution. Dermal fibroblasts, the most abundant cluster, were examined to establish a connectome with other skin cells. Annexin1-FPR1 signaling pathway was dominant in both FS as well as HGG fibroblasts and related myeloid cells while scanty in MS fibroblasts. Myeloid-specific FPR1-ORF delivered in murine wound edge using tissue nanotransfection (TNT) technology significantly enhanced the quality of healing. Pseudotime analyses identified the co-existence of an HGG fibroblast subset with FPR1high myeloid cells of fetal origin indicating common underlying biological processes.
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    Identification of a physiologic vasculogenic fibroblast state to achieve tissue repair
    (Springer Nature, 2023-02-28) Pal, Durba; Ghatak, Subhadip; Singh, Kanhaiya; Abouhashem, Ahmed Safwat; Kumar, Manishekhar; El Masry, Mohamed S.; Mohanty, Sujit K.; Palakurti, Ravichand; Rustagi, Yashika; Tabasum, Saba; Khona, Dolly K.; Khanna, Savita; Kacar, Sedat; Srivastava, Rajneesh; Bhasme, Pramod; Verma, Sumit S.; Hernandez, Edward; Sharma, Anu; Reese, Diamond; Verma, Priyanka; Ghosh, Nandini; Gorain, Mahadeo; Wan, Jun; Liu, Sheng; Liu, Yunlong; Castro, Natalia Higuita; Gnyawali, Surya C.; Lawrence, William; Moore, Jordan; Perez, Daniel Gallego; Roy, Sashwati; Yoder, Mervin C.; Sen, Chandan K.; Surgery, School of Medicine
    Tissue injury to skin diminishes miR-200b in dermal fibroblasts. Fibroblasts are widely reported to directly reprogram into endothelial-like cells and we hypothesized that miR-200b inhibition may cause such changes. We transfected human dermal fibroblasts with anti-miR-200b oligonucleotide, then using single cell RNA sequencing, identified emergence of a vasculogenic subset with a distinct fibroblast transcriptome and demonstrated blood vessel forming function in vivo. Anti-miR-200b delivery to murine injury sites likewise enhanced tissue perfusion, wound closure, and vasculogenic fibroblast contribution to perfused vessels in a FLI1 dependent manner. Vasculogenic fibroblast subset emergence was blunted in delayed healing wounds of diabetic animals but, topical tissue nanotransfection of a single anti-miR-200b oligonucleotide was sufficient to restore FLI1 expression, vasculogenic fibroblast emergence, tissue perfusion, and wound healing. Augmenting a physiologic tissue injury adaptive response mechanism that produces a vasculogenic fibroblast state change opens new avenues for therapeutic tissue vascularization of ischemic wounds.
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    Vasculogenic skin reprogramming requires TET-mediated gene demethylation in fibroblasts for rescuing impaired perfusion in diabetes
    (Springer Nature, 2024-11-27) Mohanty, Sujit K.; Singh, Kanhaiya; Kumar, Manishekhar; Verma, Sumit S.; Srivastava, Rajneesh; Gnyawali, Surya C.; Palakurti, Ravichand; Sahi, Ajay K.; El Masry, Mohamed S.; Banerjee, Pradipta; Kacar, Sedat; Rustagi, Yashika; Verma, Priyanka; Ghatak, Subhadip; Hernandez, Edward; Rubin, J. Peter; Khanna, Savita; Roy, Sashwati; Yoder, Mervin C.; Sen, Chandan K.; Surgery, School of Medicine
    Tissue nanotransfection (TNT) topically delivers Etv2, Foxc2, and Fli1 (EFF) plasmids increasing vasculogenic fibroblasts (VF) and promoting vascularization in ischemic murine skin. Human dermal fibroblasts respond to EFF nanoelectroporation with elevated expression of endothelial genes in vitro, which is linked to increased ten-eleven translocase 1/2/3 (TET) expression. Single cell RNA sequencing dependent validation of VF induction reveals a TET-dependent transcript signature. TNTEFF also induces TET expression in vivo, and fibroblast-specific EFF overexpression leads to VF-transition, with TET-activation correlating with higher 5-hydroxymethylcytosine (5-hmC) levels in VF. VF emergence requires TET-dependent demethylation of endothelial genes in vivo, enhancing VF abundance and restoring perfusion in diabetic ischemic limbs. TNTEFF improves perfusion and wound closure in diabetic mice, while increasing VF in cultured human skin explants. Suppressed in diabetes, TET1/2/3 play a critical role in TNT-mediated VF formation which supports de novo blood vessel development to rescue diabetic ischemic tissue.