Browsing by Author "Gnyawali, Surya C."
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Item 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 MedicineEpithelial 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.Item High resolution ultrasound imaging for repeated measure of wound tissue morphometry, biomechanics and hemodynamics under fetal, adult and diabetic conditions(PLOS, 2020-11-23) Gnyawali, Surya C.; Sinha, Mithun; El Masry, Mohamed S.; Wulff, Brian; Ghatak, Subhadip; Soto-Gonzalez, Fidel; Wilgus, Traci A.; Roy, Sashwati; Sen, Chandan K.; Surgery, School of MedicineNon-invasive, repeated interrogation of the same wound is necessary to understand the tissue repair continuum. In this work, we sought to test the significance of non-invasive high-frequency high-resolution ultrasound technology for such interrogation. High-frequency high-resolution ultrasound imaging was employed to investigate wound healing under fetal and adult conditions. Quantitative tissue cellularity and elastic strain was obtained for visualization of unresolved inflammation using Vevo strain software. Hemodynamic properties of the blood flow in the artery supplying the wound-site were studied using color Doppler flow imaging. Non-invasive monitoring of fetal and adult wound healing provided unprecedented biomechanical and functional insight. Fetal wounds showed highly accelerated closure with transient perturbation of wound tissue cellularity. Fetal hemodynamics was unique in that sharp fall in arterial pulse pressure (APP) which was rapidly restored within 48h post-wounding. In adults, APP transiently increased post-wounding before returning to the pre-wounding levels by d10 post-wounding. The pattern of change in the elasticity of wound-edge tissue of diabetics was strikingly different. Severe strain acquired during the early inflammatory phase persisted with a slower recovery of elasticity compared to that of the non-diabetic group. Wound bed of adult diabetic mice (db/db) showed persistent hypercellularity compared to littermate controls (db/+) indicative of prolonged inflammation. Normal skin strain of db/+ and db/db were asynchronous. In db/db, severe strain acquired during the early inflammatory phase persisted with a slower recovery of elasticity compared to that of non-diabetics. This study showcases a versatile clinically relevant imaging platform suitable for real-time analyses of functional wound healing.Item 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 MedicineTissue 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.Item Inducible miR-1224 silences cerebrovascular Serpine1 and restores blood flow to the stroke-affected site of the brain(Elsevier, 2023-01-02) Palakurti, Ravichand; Biswas, Nirupam; Roy, Sashwati; Gnyawali, Surya C.; Sinha, Mithun; Singh, Kanhaiya; Ghatak, Subhadip; Sen, Chandan K.; Khann, Savita; Surgery, School of MedicineThe α-tocotrienol (TCT) form of natural vitamin E is more potent than the better known α-tocopherol against stroke. Angiographic studies of canine stroke have revealed beneficial cerebrovascular effects of TCT. This work seeks to understand the molecular basis of such effect. In mice, TCT supplementation improved perfusion at the stroke-affected site by inducing miR-1224. miRNA profiling of a laser-capture-microdissected stroke-affected brain site identified miR-1224 as the only vascular miR induced. Lentiviral knockdown of miR-1224 significantly blunted the otherwise beneficial effects of TCT on stroke outcomes. Studies on primary brain microvascular endothelial cells revealed direct angiogenic properties of miR-1224. In mice not treated with TCT, advance stereotaxic delivery of an miR-1224 mimic to the stroke site markedly improved stroke outcomes. Mechanistic studies identified Serpine1 as a target of miR-1224. Downregulation of Serpine1 augmented the angiogenic response of the miR-1224 mimic in the brain endothelial cells. The inhibition of Serpine1, by dietary TCT and pharmacologically, increased cerebrovascular blood flow at the stroke-affected site and protected against stroke. This work assigns Serpine1, otherwise known to be of critical significance in stroke, a cerebrovascular function that worsens stroke outcomes. miR-1224-dependent inhibition of Serpine1 can be achieved by dietary TCT as well as by the small-molecule inhibitor TM5441.Item Moisture mitigation using a vented liner and a vented socket system for individuals with transfemoral amputation(Springer Nature, 2023-10-02) Gnyawali, Surya C.; Denune, Jeffrey A.; Hockman, Bryce; Valgerður Kristjánsdóttir, Jóna; Sól Ragnarsdóttir, Margrét; Timsina, Lava R.; Ghatak, Subhadip; Lechler, Knut; Sen, Chandan K.; Roy, Sashwati; Surgery, School of MedicineSweating and heat buildup at the skin-liner interface is a major challenge for persons with limb loss. Liners made of heat-non-conducting materials may cause sweating of the residual limb and may result in liners slipping off the skin surface especially on a warm day or during high activity, causing skin breakdown and affecting limb health. To address this, we evaluated the efficacy of the vented liner-socket system (VS, Össur) compared to Seal-In silicone liner and non-vented socket (nVS, Össur) in reducing relative humidity (RH) during increased sweat. Nine individuals with limb loss using nVS were randomized to VS or nVS and asked for activity in a 20-min treadmill walk. RH was significantly attenuated (p = 0.0002) and perceived sweating, as reported by prosthesis users, improved (p = 0.028) with VS, patient-reported comprehensive lower limb amputee socket survey (CLASS) outcomes to determine the suspension, stability, and comfort were not significantly different between VS and nVS. There are limited rigorous scientific studies that clearly provide evidence-based guidelines to the prosthetist in the selection of liners from numerous available options. The present study is innovative in clearly establishing objective measures for assessing humidity and temperatures at the skin-liner interface while performing activity. As shown by the measured data and perceived sweat scores provided by the subjects based on their daily experience, this study provided clear evidence establishing relative humidity at the skin-liner interface is reduced with the use of a vented liner-socket system when compared to a similar non-vented system.Item Robust critical limb ischemia porcine model involving skeletal muscle necrosis(Springer Nature, 2023-07-18) El Masry, Mohamed S.; Gnyawali, Surya C.; Sen, Chandan K.; Surgery, School of MedicineThis work sought to develop a robust and clinically relevant swine model of critical limb ischemia (CLI) involving the onset of ischemic muscle necrosis. CLI carries about 25-40% risk of major amputation with 20% annual mortality. Currently, there is no specific treatment that targets the ischemic myopathy characteristic of CLI. Current swine models of CLI, with tolerable side-effects, fail to achieve sustained ischemia followed by a necrotic myopathic endpoint. Such limitation in experimental model hinders development of effective interventions. CLI was induced unilaterally by ligation-excision of one inch of the common femoral artery (CFA) via infra-inguinal minimal incision in female Yorkshire pigs (n = 5). X-ray arteriography was done pre- and post-CFA transection to validate successful induction of severe ischemia. Weekly assessment of the sequalae of ischemia on limb perfusion, and degree of ischemic myopathy was conducted for 1 month using X-ray arteriography, laser speckle imaging, CTA angiography, femoral artery duplex, high resolution ultrasound and histopathological analysis. The non-invasive tissue analysis of the elastography images showed specific and characteristic pattern of increased muscle stiffness indicative of the fibrotic and necrotic outcome expected with associated total muscle ischemia. The prominent onset of skeletal muscle necrosis was evident upon direct inspection of the affected tissues. Ischemic myopathic changes associated with inflammatory infiltrates and deficient blood vessels were objectively validated. A translational model of severe hindlimb ischemia causing ischemic myopathy was successfully established adopting an approach that enables long-term survival studies in compliance with regulatory requirements pertaining to animal welfare.Item Skin Transcriptome of Middle-Aged Women Supplemented With Natural Herbo-mineral Shilajit Shows Induction of Microvascular and Extracellular Matrix Mechanisms(Taylor & Francis, 2019-06-04) Das, Amitava; Masry, Mohamed S. El; Gnyawali, Surya C.; Ghatak, Subhadip; Singh, Kanhaiya; Stewart, Richard; Lewis, Madeline; Saha, Abhijoy; Gordillo, Gayle; Khanna, Savita; Surgery, School of MedicineObjective: Shilajit is a pale-brown to blackish-brown organic mineral substance available from Himalayan rocks. We demonstrated that in type I obese humans, shilajit supplementation significantly upregulated extracellular matrix (ECM)–related genes in the skeletal muscle. Such an effect was highly synergistic with exercise. The present study (clinicaltrials.gov ) aimed to evaluate the effects of shilajit supplementation on skin gene expression profile and microperfusion in healthy adult females. Methods: The study design comprised six total study visits including a baseline visit (V1) and a final 14-week visit (V6) following oral shilajit supplementation (125 or 250 mg bid). A skin biopsy of the left inner upper arm of each subject was collected at visit 2 and visit 6 for gene expression profiling using Affymetrix Clariom™ D Assay. Skin perfusion was determined by MATLAB processing of dermascopic images. Transcriptome data were normalized and subjected to statistical analysis. The differentially regulated genes were subjected to Ingenuity Pathway Analysis (IPA®). The expression of the differentially regulated genes identified by IPA® were verified using real-time polymerasechain reaction (RT-PCR). Results: Supplementation with shilajit for 14 weeks was not associated with any reported adverse effect within this period. At a higher dose (250 mg bid), shilajit improved skin perfusion when compared to baseline or the placebo. Pathway analysis identified shilajit-inducible genes relevant to endothelial cell migration, growth of blood vessels, and ECM which were validated by quantitative real-time polymerasechain reaction (RT-PCR) analysis. Conclusions: This work provides maiden evidence demonstrating that oral shilajit supplementation in adult healthy women induced genes relevant to endothelial cell migration and growth of blood vessels. Shilajit supplementation improved skin microperfusion.Item 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 MedicineTissue 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.