Browsing by Author "Kumar, Manishekhar"

Now showing 1 - 9 of 9
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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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    Hydrolyzed Collagen Powder Dressing Improves Wound Inflammation, Perfusion, and Breaking Strength of Repaired Tissue
    (Liebert, 2023) Kumar, Manishekhar; Banerjee, Pradipta; Das, Amitava; Singh, Kanhaiya; Guith, Tanner; Kacar, Sedat; Gourishetti, Karthik; Sen, Chandan K.; Roy, Sashwati; Khanna, Savita; Surgery, School of Medicine
    Hydrolyzed collagen-based matrices are widely used as wound care dressings. Information on the mechanism of action of such dressings is scanty. The objective of this study was to test the effect of a specific hydrolyzed collagen powder (HCP), which is extensively used for wound care management in the United States. The effects of HCP on resolution of wound inflammation, perfusion, closure, and breaking strength of the repaired skin were studied in an experimental murine model. In early (day 7) inflammatory phase of wound macrophages, HCP treatment boosted phagocytosis and efferocytosis of wound-site macrophages. In these cells, inducible reactive oxygen species were also higher on day (d) 7. HCP treatment potentiated the expression of anti-inflammatory interleukin (IL)-10 cytokine and proangiogenic vascular endothelial growth factor (VEGF) production. Excisional wounds dressed with HCP showed complete closure on day 21, while the control wounds remained open. HCP treatment also demonstrated improved quality of wound healing as marked by the improved breaking strength of the closed wound tissue/repaired skin. These data represent first evidence on the mechanism of action of clinically used HCP. HCP dressing favorably influenced both wound inflammation and vascularization. Improved breaking strength of HCP-treated repaired skin lays the rationale for future studies testing the hypothesis that HCP-treated closed wounds would show fewer recurrences.
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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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    Mission SpaceX CRS-19 RRRM-1 space flight induced skin genomic plasticity via an epigenetic trigger
    (Elsevier, 2024-11-14) Singh, Kanhaiya; Verma, Priyanka; Srivastava, Rajneesh; Rustagi, Yashika; Kumar, Manishekhar; Verma, Sumit S.; Mohanty, Sujit; Beheshti, Afshin; Warren, Liz; Sen, Chandan K.; Surgery, School of Medicine
    Genomic plasticity helps adapt to extreme environmental conditions. We tested the hypothesis that exposure to space environment (ESE) impacts the epigenome inducing genomic plasticity. Murine skin samples from the Rodent Research Reference Mission-1 were procured from the International Space Station (ISS) National Laboratory. Targeted RNA sequencing to test differential gene expression between the skin of ESE versus ground controls revealed upregulation of VEGF-mediated angiogenesis pathways secondary to promoter hypomethylation in responders. Methylome sequencing identified ESE-sensitive hypomethylated genes including developmental angiogenic genes Araf, Vegfb, and Vegfr1. Based on differentially expressed genes, the angiogenesis biofunction was enriched in responders. The induction of genomic plasticity in response to ESE, as reported herein, may be viewed as a mark of biological resilience that is evident in a minority of organisms, responders but not in non-responders, exposed to the same stressor. Inducible genomic plasticity may be implicated in natural resilience to ESE.
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    Tissue nanotransfection causes tumor regression by its effect on nanovesicle cargo that alters microenvironmental macrophage state
    (Elsevier, 2023) Gordillo, Gayle M.; Guda, Poornachander Reddy; Singh, Kanhaiya; Biswas, Ayan; Abouhashem, Ahmed S.; Rustagi, Yashika; Sen, Abhishek; Kumar, Manishekhar; Das, Amitava; Ghatak, Subhadip; Khanna, Savita; Sen, Chandan K.; Roy, Sashwati; Surgery, School of Medicine
    Extracellular vesicles (EVs) are nanovesicles released by all eukaryotic cells. This work reports the first nanoscale fluorescent visualization of tumor-originating vesicles bearing an angiogenic microRNA (miR)-126 cargo. In a validated experimental model of lethal murine vascular neoplasm, tumor-originating EV delivered its miR-126 cargo to tumor-associated macrophages (TAMs). Such delivery resulted in an angiogenic (LYVE+) change of state in TAM that supported tumor formation. Study of the trafficking of tumor-originating fluorescently tagged EV revealed colocalization with TAM demonstrating uptake by these cells. Ex vivo treatment of macrophages with tumor-derived EVs led to gain of tumorigenicity in these isolated cells. Single-cell RNA sequencing of macrophages revealed that EV-borne miR-126 characterized the angiogenic change of state. Unique gene expression signatures of specific macrophage clusters responsive to miR-126-enriched tumor-derived EVs were revealed. Topical tissue nanotransfection (TNT) delivery of an oligonucleotide comprising an anti-miR against miR-126 resulted in significant knockdown of miR-126 in the tumor tissue. miR-126 knockdown resulted in complete involution of the tumor and improved survival rate of tumor-affected mice. This work identifies a novel tumorigenic mechanism that relies on tumorigenic state change of TAM caused by tumor-originating EV-borne angiomiR. This disease process can be effectively targeted by topical TNT of superficial tumors.
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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.