44 results on '"Alexander J. Whittam"'
Search Results
2. The Role of Focal Adhesion Kinase in Keratinocyte Fibrogenic Gene Expression
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Michael Januszyk, Sun Hyung Kwon, Victor W. Wong, Jagannath Padmanabhan, Zeshaan N. Maan, Alexander J. Whittam, Melanie R. Major, and Geoffrey C. Gurtner
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focal adhesion kinase ,keratinocyte ,mechanotransduction ,extracellular matrix ,single-cell transcriptional analysis ,skin fibrosis ,hypertrophic scar ,transcriptomics ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Abnormal skin scarring causes functional impairment, psychological stress, and high socioeconomic cost. Evidence shows that altered mechanotransduction pathways have been linked to both inflammation and fibrosis, and that focal adhesion kinase (FAK) is a key mediator of these processes. We investigated the importance of keratinocyte FAK at the single cell level in key fibrogenic pathways critical for scar formation. Keratinocytes were isolated from wildtype and keratinocyte-specific FAK-deleted mice, cultured, and sorted into single cells. Keratinocytes were evaluated using a microfluidic-based platform for high-resolution transcriptional analysis. Partitive clustering, gene enrichment analysis, and network modeling were applied to characterize the significance of FAK on regulating keratinocyte subpopulations and fibrogenic pathways important for scar formation. Considerable transcriptional heterogeneity was observed within the keratinocyte populations. FAK-deleted keratinocytes demonstrated increased expression of genes integral to mechanotransduction and extracellular matrix production, including Igtbl, Mmpla, and Col4a1. Transcriptional activities upon FAK deletion were not identical across all single keratinocytes, resulting in higher frequency of a minor subpopulation characterized by a matrix-remodeling profile compared to wildtype keratinocyte population. The importance of keratinocyte FAK signaling gene expression was revealed. A minor subpopulation of keratinocytes characterized by a matrix-modulating profile may be a keratinocyte subset important for mechanotransduction and scar formation.
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- 2017
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3. Optimization of transdermal deferoxamine leads to enhanced efficacy in healing skin wounds
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Clark A. Bonham, Alexander J. Whittam, Dominik Duscher, Jayakumar Rajadas, Geoffrey C. Gurtner, Karl Engel, Zachary A. Stern-Buchbinder, Artem A. Trotsyuk, Mohammed Inayathullah, Janos A. Barrera, Michael S. Hu, Zeshaan N. Maan, Melanie Rodrigues, and Sun Hyung Kwon
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Male ,Chronic wound ,Neovascularization, Physiologic ,Siderophores ,Pharmaceutical Science ,02 engineering and technology ,Deferoxamine ,Pharmacology ,Administration, Cutaneous ,Neovascularization ,Mice ,03 medical and health sciences ,Drug Delivery Systems ,Animals ,Humans ,Medicine ,Adverse effect ,Skin ,030304 developmental biology ,Transdermal ,Wound Healing ,0303 health sciences ,business.industry ,Regeneration (biology) ,021001 nanoscience & nanotechnology ,Mice, Inbred C57BL ,Drug Liberation ,Drug delivery ,Collagen ,medicine.symptom ,0210 nano-technology ,business ,Wound healing ,medicine.drug - Abstract
Chronic wounds remain a significant burden to both the healthcare system and individual patients, indicating an urgent need for new interventions. Deferoxamine (DFO), an iron-chelating agent clinically used to treat iron toxicity, has been shown to reduce oxidative stress and increase hypoxia-inducible factor-1 alpha (HIF-1α) activation, thereby promoting neovascularization and enhancing regeneration in chronic wounds. However due to its short half-life and adverse side effects associated with systemic absorption, there is a pressing need for targeted DFO delivery. We recently published a preclinical proof of concept drug delivery system (TDDS) which showed that transdermally applied DFO is effective in improving chronic wound healing. Here we present an enhanced TDDS (eTDDS) comprised exclusively of FDA-compliant constituents to optimize drug release and expedite clinical translation. We evaluate the eTDDS to the original TDDS and compare this with other commonly used delivery methods including DFO drip-on and polymer spray applications. The eTDDS displayed excellent physicochemical characteristics and markedly improved DFO delivery into human skin when compared to other topical application techniques. We demonstrate an accelerated wound healing response with the eTDDS treatment resulting in significantly increased wound vascularity, dermal thickness, collagen deposition and tensile strength. Together, these findings highlight the immediate clinical potential of DFO eTDDS to treating diabetic wounds. Further, the topical drug delivery platform has important implications for targeted pharmacologic therapy of a wide range of cutaneous diseases.
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- 2019
4. Small molecule inhibition of dipeptidyl peptidase-4 enhances bone marrow progenitor cell function and angiogenesis in diabetic wounds
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Zeshaan N. Maan, Dominik Duscher, Janos A. Barrera, Victor W. Wong, Sacha M.L. Khong, Ferdinando Giacco, Michael T. Longaker, Michael Brownlee, Michael Januszyk, Michael S. Hu, Sun Hyung Kwon, Graham G. Walmsley, Lauren H. Fischer, Geoffrey C. Gurtner, and Alexander J. Whittam
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0301 basic medicine ,Stromal cell ,Angiogenesis ,Dipeptidyl Peptidase 4 ,Population ,Article ,Diabetes Mellitus, Experimental ,Neovascularization ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Physiology (medical) ,Animals ,Medicine ,Progenitor cell ,education ,Dipeptidyl peptidase-4 ,Dipeptidyl-Peptidase IV Inhibitors ,Wound Healing ,education.field_of_study ,Neovascularization, Pathologic ,business.industry ,Biochemistry (medical) ,Public Health, Environmental and Occupational Health ,General Medicine ,Triazoles ,Hematopoietic Stem Cells ,Chemokine CXCL12 ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Cancer research ,Wounds and Injuries ,Bone marrow ,medicine.symptom ,business ,Wound healing ,Glipizide - Abstract
In diabetes, stromal cell-derived factor-1 (SDF-1) expression and progenitor cell recruitment are reduced. Dipeptidyl peptidase-4 (DPP-4) inhibits SDF-1 expression and progenitor cell recruitment. Here we examined the impact of the DPP-4 inhibitor, MK0626, on progenitor cell kinetics in the context of wound healing. Wildtype (WT) murine fibroblasts cultured under high-glucose to reproduce a diabetic microenvironment were exposed to MK0626, glipizide, or no treatment, and SDF-1 expression was measured with ELISA. Diabetic mice received MK0626, glipizide, or no treatment for 6 weeks and then were wounded. Immunohistochemistry was used to quantify neovascularization and SDF-1 expression. Gene expression was measured at the RNA and protein level using quantitative polymerase chain reaction and ELISA, respectively. Flow cytometry was used to characterize bone marrow-derived mesenchymal progenitor cell (BM-MPC) population recruitment to wounds. BM-MPC gene expression was assayed using microfluidic single cell analysis. WT murine fibroblasts exposed to MK0626 demonstrated increased SDF-1 expression. MK0626 treatment significantly accelerated wound healing and increased wound vascularity, SDF-1 expression, and dermal thickness in diabetic wounds. MK0626 treatment increased the number of BM-MPCs present in bone marrow and in diabetic wounds. MK0626 had no effect on BM-MPC population dynamics. BM-MPCs harvested from MK0626-treated mice exhibited increased chemotaxis in response to SDF-1 when compared to diabetic controls. Treatment with a DPP-4 inhibitor significantly improved wound healing, angiogenesis, and endogenous progenitor cell recruitment in the setting of diabetes.
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- 2019
5. Endothelial Cxcl12 Regulates Neovascularization During Tissue Repair and Tumor Progression
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Geoffrey C. Gurtner, Lauren H. Fischer, Robert C. Rennert, Dharshan Sivaraj, Michael T. Longaker, Michael Januszyk, Zeshaan N. Maan, Alexander J. Whittam, Jagannath Padmanabhan, Dominic Henn, Kellen Chen, Wing Lam Natalie Ho, Johannes Riegler, Ivan N. Vial, Joseph C. Wu, Janos A. Barrera, Michael S. Hu, and Dominik Duscher
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Stromal cell ,Parabiosis ,Biology ,biological factors ,Neovascularization ,Haematopoiesis ,medicine.anatomical_structure ,Tumor progression ,embryonic structures ,Gene expression ,Knockout mouse ,medicine ,Cancer research ,biological phenomena, cell phenomena, and immunity ,medicine.symptom ,Fibroblast - Abstract
CXC chemokine ligand 12 (CXCL12; stromal cell-derived factor 1 [SDF-1]), primarily known for its role in embryogenesis and hematopoiesis, has also been implicated in tumor biology and neovascularization. However, its specific role and mechanism of action remain poorly understood. We previously demonstrated that CXCL12 expression is Hypoxia-Inducible Factor (HIF)-1 responsive. Here we use a conditional CXCL12 knockout mouse to show that endothelial-specific deletion of CXCL12 (eKO) does not affect embryogenesis, but reduces the survival of ischemic tissue, altering tissue repair and tumor progression. Loss of vascular endothelial CXCL12 disrupts endothelial – fibroblast crosstalk necessary for stromal growth and vascularization. Single-cell gene expression analysis in combination with a parabiosis model reveals a specific population of non-inflammatory circulating cells, defined by genes regulating neovascularization, which is recruited by endothelial CXCL12. These findings indicate an essential role for endothelial CXCL12 expression during the adult neovascular response in tissue injury and tumor progression.
- Published
- 2021
6. Endothelial CXCL12 regulates neovascularization during tissue repair and tumor progression
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Robert C. Rennert, Zeshaan N. Maan, Michael S. Hu, Michael T. Longaker, Johannes Riegler, Jagannath Padmanabhan, Natalie Ho, Dominik Duscher, Geoffrey C. Gurtner, Michael Januszyk, Alexander J. Whittam, Joseph C. Wu, Janos A. Barrera, Lauren H. Fischer, and Ivan N. Vial
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Stromal cell ,Parabiosis ,Biology ,biological factors ,Neovascularization ,Haematopoiesis ,medicine.anatomical_structure ,Tumor progression ,embryonic structures ,Knockout mouse ,Gene expression ,medicine ,Cancer research ,biological phenomena, cell phenomena, and immunity ,medicine.symptom ,Fibroblast - Abstract
CXC chemokine ligand 12 (CXCL12; stromal cell-derived factor 1 [SDF-1]), primarily known for its role in embryogenesis and hematopoiesis, has also been implicated in tumor biology and neovascularization. However, its specific role and mechanism of action remain poorly understood. We previously demonstrated that CXCL12 expression is Hypoxia-Inducible Factor (HIF)-1 responsive. Here we use a conditional CXCL12 knockout mouse to show that endothelial-specific deletion of CXCL12 (eKO) does not affect embryogenesis, but reduces the survival of ischemic tissue, altering tissue repair and tumor progression. Loss of vascular endothelial CXCL12 disrupts endothelial – fibroblast crosstalk necessary for stromal growth and vascularization. Single-cell gene expression analysis in combination with a parabiosis model reveals a specific population of non-inflammatory circulating cells, defined by genes regulating neovascularization, which is recruited by endothelial CXCL12. These findings indicate an essential role for endothelial CXCL12 expression during the adult neovascular response in tissue injury and tumor progression.
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- 2020
7. Comparison of the Hydroxylase Inhibitor Dimethyloxalylglycine and the Iron Chelator Deferoxamine in Diabetic and Aged Wound Healing
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Sacha M.L. Khong, Michael T. Longaker, Yixiao Dong, Graham G. Walmsley, Geoffrey C. Gurtner, Alexander J. Whittam, Michael S. Hu, Dominik Duscher, Zeshaan N. Maan, and Michael Januszyk
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0301 basic medicine ,Iron Chelator ,Pathology ,medicine.medical_specialty ,business.industry ,medicine.disease ,Deferoxamine ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Endocrinology ,030220 oncology & carcinogenesis ,Diabetes mellitus ,Internal medicine ,medicine ,Surgery ,Wound healing ,business ,Transcription factor ,medicine.drug - Abstract
Background:A hallmark of diabetes mellitus is the breakdown of almost every reparative process in the human body, leading to critical impairments of wound healing. Stabilization and activity of the transcription factor hypoxia-inducible factor (HIF)-1α is impaired in diabetes, leading to deficits in
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- 2017
8. Extracellular superoxide dismutase deficiency impairs wound healing in advanced age by reducing neovascularization and fibroblast function
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Toshihiro Fujiwara, Kristine C. Rustad, Geoffrey C. Gurtner, Michael Januszyk, Revanth Kosaraju, Melanie Rodrigues, Alexander J. Whittam, Zeshaan N. Maan, and Dominik Duscher
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Male ,0301 basic medicine ,Aging ,Neutrophils ,SOD3 ,Neovascularization, Physiologic ,Dermatology ,medicine.disease_cause ,Biochemistry ,Article ,Antioxidants ,Transforming Growth Factor beta1 ,Superoxide dismutase ,Neovascularization ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Extracellular ,medicine ,Animals ,Fibroblast ,Molecular Biology ,Cell Proliferation ,Mice, Knockout ,chemistry.chemical_classification ,Wound Healing ,Reactive oxygen species ,biology ,Superoxide Dismutase ,business.industry ,Fibroblasts ,Cell biology ,Mice, Inbred C57BL ,Oxygen ,Oxidative Stress ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,030220 oncology & carcinogenesis ,Mutation ,Immunology ,biology.protein ,medicine.symptom ,Reactive Oxygen Species ,Wound healing ,business ,Oxidative stress - Abstract
Advanced age is characterized by impairments in wound healing, and evidence is accumulating that this may be due in part to a concomitant increase in oxidative stress. Extended exposure to reactive oxygen species (ROS) is thought to lead to cellular dysfunction and organismal death via the destructive oxidation of intra-cellular proteins, lipids and nucleic acids. Extracellular superoxide dismutase (ecSOD/SOD3) is a prime antioxidant enzyme in the extracellular space that eliminates ROS. Here, we demonstrate that reduced SOD3 levels contribute to healing impairments in aged mice. These impairments include delayed wound closure, reduced neovascularization, impaired fibroblast proliferation and increased neutrophil recruitment. We further establish that SOD3 KO and aged fibroblasts both display reduced production of TGF-β1, leading to decreased differentiation of fibroblasts into myofibroblasts. Taken together, these results suggest that wound healing impairments in ageing are associated with increased levels of ROS, decreased SOD3 expression and impaired extracellular oxidative stress regulation. Our results identify SOD3 as a possible target to correct age-related cellular dysfunction in wound healing.
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- 2016
9. Challenges and Opportunities in Drug Delivery for Wound Healing
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Geoffrey C. Gurtner, Michael Januszyk, Alexander J. Whittam, Dominik Duscher, Zeshaan N. Maan, Janos A. Barrera, and Victor W. Wong
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0301 basic medicine ,Skin barrier ,integumentary system ,business.industry ,Tissue repair ,Critical Care and Intensive Care Medicine ,Bioinformatics ,Regenerative medicine ,030207 dermatology & venereal diseases ,03 medical and health sciences ,Wound care ,030104 developmental biology ,0302 clinical medicine ,Impaired tissue repair ,Physical Barrier ,Drug delivery ,Emergency Medicine ,Medicine ,business ,Wound healing ,Critical Reviews - Abstract
Significance: Chronic wounds remain a significant public health problem. Alterations in normal physiological processes caused by aging or diabetes lead to impaired tissue repair and the development of chronic and nonhealing wounds. Understanding the unique features of the wound environment will be required to develop new therapeutics that impact these disabling conditions. New drug-delivery systems (DDSs) may enhance current and future therapies for this challenging clinical problem. Recent Advances: Historically, physical barriers and biological degradation limited the efficacy of DDSs in wound healing. In aiming at improving and optimizing drug delivery, recent data suggest that combinations of delivery mechanisms, such as hydrogels, small molecules, RNA interference (RNAi), as well as growth factor and stem cell-based therapies (biologics), could offer exciting new opportunities for improving tissue repair. Critical Issues: The lack of effective therapeutic approaches to combat the significant disability associated with chronic wounds has become an area of increasing clinical concern. However, the unique challenges of the wound environment have limited the development of effective therapeutic options for clinical use. Future Directions: New platforms presented in this review may provide clinicians and scientists with an improved understanding of the alternatives for drug delivery in wound care, which may facilitate the development of new therapeutic approaches for patients.
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- 2016
10. Cell-Assisted Lipotransfer Improves Volume Retention in Irradiated Recipient Sites and Rescues Radiation-Induced Skin Changes
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Anna Luan, Derrick C. Wan, Gordon K. Lee, Elizabeth R. Zielins, Dominik Duscher, Geoffrey C. Gurtner, Alexander J. Whittam, Kevin J. Paik, Elizabeth A. Brett, Michael T. Longaker, Michael S. Hu, and David Atashroo
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Pathology ,medicine.medical_specialty ,Stromal cell ,medicine.medical_treatment ,030230 surgery ,Biology ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Vascularity ,Fibrosis ,Adipocytes ,medicine ,Animals ,Humans ,Subcutaneous fibrosis ,Skin ,Radiotherapy ,Graft Survival ,Soft tissue ,Cell Biology ,Anatomy ,Stromal vascular fraction ,medicine.disease ,Radiation therapy ,030220 oncology & carcinogenesis ,Microvessels ,Molecular Medicine ,Stromal Cells ,Stem cell ,medicine.symptom ,Developmental Biology - Abstract
Radiation therapy is not only a mainstay in the treatment of many malignancies but also results in collateral obliteration of microvasculature and dermal/subcutaneous fibrosis. Soft tissue reconstruction of hypovascular, irradiated recipient sites through fat grafting remains challenging; however, a coincident improvement in surrounding skin quality has been noted. Cell-assisted lipotransfer (CAL), the enrichment of fat with additional adipose-derived stem cells (ASCs) from the stromal vascular fraction, has been shown to improve fat volume retention, and enhanced outcomes may also be achieved with CAL at irradiated sites. Supplementing fat grafts with additional ASCs may also augment the regenerative effect on radiation-damaged skin. In this study, we demonstrate the ability for CAL to enhance fat graft volume retention when placed beneath the irradiated scalps of immunocompromised mice. Histologic metrics of fat graft survival were also appreciated, with improved structural qualities and vascularity. Finally, rehabilitation of radiation-induced soft tissue changes were also noted, as enhanced amelioration of dermal thickness, collagen content, skin vascularity, and biomechanical measures were all observed with CAL compared to unsupplemented fat grafts. Supplementation of fat grafts with ASCs therefore shows promise for reconstruction of complex soft tissue defects following adjuvant radiotherapy.
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- 2016
11. Ultrasound-Assisted Liposuction Does Not Compromise the Regenerative Potential of Adipose-Derived Stem Cells
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Michael S. Pollhammer, David Atashroo, Alexander J. Whittam, Zeshaan N. Maan, Hans-Günther Machens, Elizabeth R. Zielins, Michael S. Hu, Manfred Schmidt, Georg M. Huemer, Anna Luan, Janos A. Barrera, Graham G. Walmsley, Dominik Duscher, Derrick C. Wan, Sacha M.L. Khong, Arndt F. Schilling, Geoffrey C. Gurtner, Michael T. Longaker, and Elizabeth A. Brett
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Adipose-derived stem cells ,Male ,0301 basic medicine ,medicine.medical_treatment ,Adult mesenchymal stem cells ,Regenerative medicine ,Cell therapy ,Mice ,0302 clinical medicine ,Tissue engineering ,Adipocytes ,Ultrasonography ,Adipogenesis ,Cell Differentiation ,General Medicine ,Middle Aged ,Flow Cytometry ,3. Good health ,Cell biology ,Elective Surgical Procedures ,030220 oncology & carcinogenesis ,Intercellular Signaling Peptides and Proteins ,Female ,Adult ,medicine.medical_specialty ,Cell Survival ,Abdominal Fat ,Mice, Nude ,Neovascularization, Physiologic ,03 medical and health sciences ,Chondrocytes ,Lipectomy ,Antigens, CD ,Tissue Engineering and Regenerative Medicine ,medicine ,Animals ,Humans ,Progenitor cell ,Fat harvest ,Wound Healing ,Osteoblasts ,business.industry ,Regeneration (biology) ,Mesenchymal stem cell ,Mesenchymal Stem Cells ,Cell Biology ,Surgery ,030104 developmental biology ,Liposuction ,Ultrasound-assisted liposuction ,Wound healing ,business ,Biomarkers ,Developmental Biology - Abstract
The regenerative abilities of adipose-derived mesenchymal stem cells (ASCs) harvested via a third-generation ultrasound-assisted liposuction (UAL) device versus ASCs obtained via standard suction-assisted lipoaspiration were evaluated. ASC yield and viability, and expression of most osteogenic, adipogenic, and key regenerative genes were equivalent between the two methods. Cells harvested via UAL showed comparable abilities to enhance cutaneous regeneration and appear suitable for cell therapy and tissue engineering applications., Human mesenchymal stem cells (MSCs) have recently become a focus of regenerative medicine, both for their multilineage differentiation capacity and their excretion of proregenerative cytokines. Adipose-derived mesenchymal stem cells (ASCs) are of particular interest because of their abundance in fat tissue and the ease of harvest via liposuction. However, little is known about the impact of different liposuction methods on the functionality of ASCs. Here we evaluate the regenerative abilities of ASCs harvested via a third-generation ultrasound-assisted liposuction (UAL) device versus ASCs obtained via standard suction-assisted lipoaspiration (SAL). Lipoaspirates were sorted using fluorescent assisted cell sorting based on an established surface-marker profile (CD34+/CD31−/CD45−), to obtain viable ASCs. Yield and viability were compared and the differentiation capacities of the ASCs were assessed. Finally, the regenerative potential of ASCs was examined using an in vivo model of tissue regeneration. UAL- and SAL-derived samples demonstrated equivalent ASC yield and viability, and UAL ASCs were not impaired in their osteogenic, adipogenic, or chondrogenic differentiation capacity. Equally, quantitative real-time polymerase chain reaction showed comparable expression of most osteogenic, adipogenic, and key regenerative genes between both ASC groups. Cutaneous regeneration and neovascularization were significantly enhanced in mice treated with ASCs obtained by either UAL or SAL compared with controls, but there were no significant differences in healing between cell-therapy groups. We conclude that UAL is a successful method of obtaining fully functional ASCs for regenerative medicine purposes. Cells harvested with this alternative approach to liposuction are suitable for cell therapy and tissue engineering applications. Significance Adipose-derived mesenchymal stem cells (ASCs) are an appealing source of therapeutic progenitor cells because of their multipotency, diverse cytokine profile, and ease of harvest via liposuction. Alternative approaches to classical suction-assisted liposuction are gaining popularity; however, little evidence exists regarding the impact of different liposuction methods on the regenerative functionality of ASCs. Human ASC characteristics and regenerative capacity were assessed when harvested via ultrasound-assisted (UAL) versus standard suction-assisted liposuction. ASCs obtained via UAL were of equal quality when directly compared with the current gold standard harvest method. UAL is an adjunctive source of fully functional mesenchymal stem cells for applications in basic research and clinical therapy.
- Published
- 2015
12. Evaluating the Effect of Cell Culture on Gene Expression in Primary Tissue Samples Using Microfluidic-Based Single Cell Transcriptional Analysis
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Arnetha J. Whitmore, Zeshaan N. Maan, Michael Januszyk, Geoffrey C. Gurtner, Alexander J. Whittam, Lisa K. Wong, Robert C. Rennert, Michael Sorkin, and Dominik Duscher
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transcriptional analysis ,Future studies ,Cell ,Population ,microfluidics ,Biomedical Engineering ,Bioengineering ,Biology ,Biochemistry ,Article ,lcsh:Biochemistry ,singe-cell ,Gene expression ,medicine ,lcsh:QD415-436 ,Transcriptional analysis ,education ,subpopulations ,Healing wounds ,cell culture ,education.field_of_study ,bioinformatics ,Cell biology ,medicine.anatomical_structure ,Cell culture ,gene expression ,Stem cell ,Biotechnology - Abstract
Significant transcriptional heterogeneity is an inherent property of complex tissues such as tumors and healing wounds. Traditional methods of high-throughput analysis rely on pooling gene expression data from hundreds of thousands of cells and reporting a population-wide average that is unable to capture differences within distinct cell subsets. Recent advances in microfluidic technology have permitted the development of large-scale single cell analytic methods that overcome this limitation. The increased granularity afforded by such approaches allows us to answer the critical question of whether expansion in cell culture significantly alters the transcriptional characteristics of cells isolated from primary tissue. Here we examine an established population of human adipose-derived stem cells (ASCs) using a novel, microfluidic-based method for high-throughput transcriptional interrogation, coupled with advanced bioinformatic analysis, to evaluate the dynamics of single cell gene expression among primary, passage 0, and passage 1 stem cells. We find significant differences in the transcriptional profiles of cells from each group, as well as a considerable shift in subpopulation dynamics as those subgroups better able to adhere and proliferate under these culture conditions gradually emerge as dominant. Taken together, these findings reinforce the importance of using primary or very early passage cells in future studies.
- Published
- 2015
13. dsRNA Released by Tissue Damage Activates TLR3 to Drive Skin Regeneration
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M. Zulfiquer Hossain, Dongwon Kim, Alexander J. Whittam, Emily Chang, Lloyd S. Miller, Carly Page, Sashank Reddy, Tabetha S. Ratliff, Sydney R. Resnik, Amadeus S. Zhu, Amanda M. Nelson, Luis A. Garza, and Adiya S. Katseff
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Keratinocytes ,Male ,STAT3 Transcription Factor ,Genotype ,viruses ,Cellular differentiation ,Kruppel-Like Transcription Factors ,Zinc Finger Protein Gli2 ,Biology ,Article ,Neogenesis ,Morphogenesis ,Genetics ,medicine ,Animals ,Humans ,Regeneration ,Ectodysplasin A receptor ,Phosphorylation ,Promoter Regions, Genetic ,beta Catenin ,RNA, Double-Stranded ,Skin ,Wound Healing ,integumentary system ,Interleukin-6 ,Regeneration (biology) ,Infant, Newborn ,Wnt signaling pathway ,Cell Differentiation ,Cell Biology ,Hair follicle ,biology.organism_classification ,Toll-Like Receptor 3 ,Cell biology ,Mice, Inbred C57BL ,medicine.anatomical_structure ,TLR3 ,Immunology ,Molecular Medicine ,Female ,Wound healing ,Hair Follicle ,Biomarkers ,Signal Transduction - Abstract
SummaryRegeneration of skin and hair follicles after wounding—a process known as wound-induced hair neogenesis (WIHN)—is a rare example of adult organogenesis in mammals. As such, WIHN provides a unique model system for deciphering mechanisms underlying mammalian regeneration. Here, we show that dsRNA, which is released from damaged skin, activates Toll-Like Receptor 3 (TLR3) and its downstream effectors IL-6 and STAT3 to promote hair follicle regeneration. Conversely, TLR3-deficient animals fail to initiate WIHN. TLR3 activation promotes expression of hair follicle stem cell markers and induces elements of the core hair morphogenetic program, including ectodysplasin A receptor (EDAR) and the Wnt and Shh pathways. Our results therefore show that dsRNA and TLR3 link the earliest events of mammalian skin wounding to regeneration and suggest potential therapeutic approaches for promoting hair neogenesis.
- Published
- 2015
14. The Role of Focal Adhesion Kinase in Keratinocyte Fibrogenic Gene Expression
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Sun Hyung Kwon, Zeshaan N. Maan, Geoffrey C. Gurtner, Michael Januszyk, Alexander J. Whittam, Jagannath Padmanabhan, Victor W. Wong, and Melanie R. Major
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0301 basic medicine ,Keratinocytes ,focal adhesion kinase ,keratinocyte ,mechanotransduction ,extracellular matrix ,single-cell transcriptional analysis ,skin fibrosis ,hypertrophic scar ,transcriptomics ,Mechanotransduction, Cellular ,Extracellular matrix ,lcsh:Chemistry ,0302 clinical medicine ,Gene expression ,Mechanotransduction ,lcsh:QH301-705.5 ,Spectroscopy ,Mice, Knockout ,education.field_of_study ,General Medicine ,Computer Science Applications ,Cell biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Keratinocyte ,Signal Transduction ,Population ,Biology ,Catalysis ,Article ,Inorganic Chemistry ,Focal adhesion ,03 medical and health sciences ,Hypertrophic scar ,Mediator ,medicine ,Animals ,Humans ,Physical and Theoretical Chemistry ,education ,Molecular Biology ,Focal Adhesions ,Organic Chemistry ,medicine.disease ,030104 developmental biology ,lcsh:Biology (General) ,lcsh:QD1-999 ,Focal Adhesion Protein-Tyrosine Kinases - Abstract
Abnormal skin scarring causes functional impairment, psychological stress, and high socioeconomic cost. Evidence shows that altered mechanotransduction pathways have been linked to both inflammation and fibrosis, and that focal adhesion kinase (FAK) is a key mediator of these processes. We investigated the importance of keratinocyte FAK at the single cell level in key fibrogenic pathways critical for scar formation. Keratinocytes were isolated from wildtype and keratinocyte-specific FAK-deleted mice, cultured, and sorted into single cells. Keratinocytes were evaluated using a microfluidic-based platform for high-resolution transcriptional analysis. Partitive clustering, gene enrichment analysis, and network modeling were applied to characterize the significance of FAK on regulating keratinocyte subpopulations and fibrogenic pathways important for scar formation. Considerable transcriptional heterogeneity was observed within the keratinocyte populations. FAK-deleted keratinocytes demonstrated increased expression of genes integral to mechanotransduction and extracellular matrix production, including Igtbl, Mmpla, and Col4a1. Transcriptional activities upon FAK deletion were not identical across all single keratinocytes, resulting in higher frequency of a minor subpopulation characterized by a matrix-remodeling profile compared to wildtype keratinocyte population. The importance of keratinocyte FAK signaling gene expression was revealed. A minor subpopulation of keratinocytes characterized by a matrix-modulating profile may be a keratinocyte subset important for mechanotransduction and scar formation.
- Published
- 2017
15. Age-associated intracellular superoxide dismutase deficiency potentiates dermal fibroblast dysfunction during wound healing
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Hirotaka Suga, Geoffrey C. Gurtner, Dominik Duscher, Alexander J. Whittam, Toshihiro Fujiwara, Sun Hyung Kwon, Teruyuki Dohi, Zeshaan N. Maan, Kristine C. Rustad, Jagannath Padmanabhan, and Melanie Rodrigues
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0301 basic medicine ,Male ,Aging ,MAP Kinase Signaling System ,Dermatology ,medicine.disease_cause ,Biochemistry ,Superoxide dismutase ,Dermal fibroblast ,030207 dermatology & venereal diseases ,03 medical and health sciences ,0302 clinical medicine ,Superoxide Dismutase-1 ,medicine ,Animals ,Fibroblast ,Molecular Biology ,chemistry.chemical_classification ,Reactive oxygen species ,Wound Healing ,biology ,Cell Differentiation ,Fibroblasts ,Cell biology ,Mice, Inbred C57BL ,Oxidative Stress ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,Immunology ,biology.protein ,Wound healing ,Myofibroblast ,Oxidative stress ,Intracellular - Abstract
Reactive oxygen species (ROS) impair wound healing through destructive oxidation of intracellular proteins, lipids and nucleic acids. Intracellular superoxide dismutase (SOD1) regulates ROS levels and plays a critical role in tissue homoeostasis. Recent evidence suggests that age-associated wound healing impairments may partially result from decreased SOD1 expression. We investigated the mechanistic basis by which increased oxidative stress links to age-associated impaired wound healing. Fibroblasts were isolated from unwounded skin of young and aged mice, and myofibroblast differentiation was assessed by measuring α-smooth muscle actin and collagen gel contraction. Excisional wounds were created on young and aged mice to study the healing rate, ROS levels and SOD1 expression. A mechanistic link between oxidative stress and fibroblast function was explored by assessing the TGF-β1 signalling pathway components in young and aged mice. Age-related wounds displayed reduced myofibroblast differentiation and delayed wound healing, consistent with a decrease in the in vitro capacity for fibroblast-myofibroblast transition following oxidative stress. Young fibroblasts with normal SOD1 expression exhibited increased phosphorylation of ERK in response to elevated ROS. In contrast, aged fibroblasts with reduced SOD1 expression displayed a reduced capacity to modulate intracellular ROS. Collectively, age-associated wound healing impairments are associated with fibroblast dysfunction that is likely the result of decreased SOD1 expression and subsequent dysregulation of intracellular ROS. Strategies targeting these mechanisms may suggest a new therapeutic approach in the treatment of chronic non-healing wounds in the aged population.
- Published
- 2017
16. Ultrasound-assisted liposuction provides a source for functional adipose-derived stromal cells
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Dominik Duscher, David Atashroo, Arndt F. Schilling, Michael S. Hu, Matthias M. Aitzetmüller, Alexander J. Whittam, Michael T. Longaker, Geoffrey C. Gurtner, Graham G. Walmsley, Anna Luan, Zeshaan N. Maan, Elizabeth A. Brett, Khosrow Siamak Houschyar, Derrick C. Wan, and Hans-Guenther Machens
- Subjects
0301 basic medicine ,Adult ,Male ,Cancer Research ,Pathology ,medicine.medical_specialty ,Stromal cell ,medicine.medical_treatment ,Immunology ,CD34 ,Adipose tissue ,Mice, Nude ,Regenerative medicine ,Article ,Cell therapy ,03 medical and health sciences ,0302 clinical medicine ,Tissue engineering ,Lipectomy ,Osteogenesis ,Adipocytes ,Immunology and Allergy ,Medicine ,Animals ,Humans ,Regeneration ,Genetics (clinical) ,Ultrasonography ,Transplantation ,Wound Healing ,Adipogenesis ,business.industry ,Mesenchymal stem cell ,Cell Differentiation ,Mesenchymal Stem Cells ,Cell Biology ,Middle Aged ,Flow Cytometry ,Cell biology ,030104 developmental biology ,Oncology ,Adipose Tissue ,030220 oncology & carcinogenesis ,Liposuction ,Female ,Stromal Cells ,business ,Biomarkers - Abstract
Background aims Regenerative medicine employs human mesenchymal stromal cells (MSCs) for their multi-lineage plasticity and their pro-regenerative cytokine secretome. Adipose-derived mesenchymal stromal cells (ASCs) are concentrated in fat tissue, and the ease of harvest via liposuction makes them a particularly interesting cell source. However, there are various liposuction methods, and few have been assessed regarding their impact on ASC functionality. Here we study the impact of the two most popular ultrasound-assisted liposuction (UAL) devices currently in clinical use, VASER (Solta Medical) and Lysonix 3000 (Mentor) on ASCs. Methods After lipoaspirate harvest and processing, we sorted for ASCs using fluorescent-assisted cell sorting based on an established surface marker profile (CD34+CD31–CD45–). ASC yield, viability, osteogenic and adipogenic differentiation capacity and in vivo regenerative performance were assessed. Results Both UAL samples demonstrated equivalent ASC yield and viability. VASER UAL ASCs showed higher osteogenic and adipogenic marker expression, but a comparable differentiation capacity was observed. Soft tissue healing and neovascularization were significantly enhanced via both UAL-derived ASCs in vivo, and there was no significant difference between the cell therapy groups. Conclusions Taken together, our data suggest that UAL allows safe and efficient harvesting of the mesenchymal stromal cellular fraction of adipose tissue and that cells harvested via this approach are suitable for cell therapy and tissue engineering applications.
- Published
- 2017
17. Understanding regulatory pathways of neovascularization in diabetes
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Alexander J. Whittam, Robert C. Rennert, Michael S. Hu, Zeshaan N. Maan, Christopher R. Davis, Geoffrey C. Gurtner, Arnetha J. Whitmore, Dominik Duscher, Michael Januszyk, and Melanie Rodrigues
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medicine.medical_specialty ,Angiogenesis ,business.industry ,Endocrinology, Diabetes and Metabolism ,Diabetic retinopathy ,Disease ,Hypoxia (medical) ,medicine.disease ,Bioinformatics ,Neovascularization ,Endocrinology ,Internal medicine ,Diabetes mellitus ,medicine ,Etiology ,Myocardial infarction ,medicine.symptom ,business - Abstract
Diabetes mellitus and its associated comorbidities represent a significant health burden worldwide. Vascular dysfunction is the major contributory factor in the development of these comorbidities, which include impaired wound healing, cardiovascular disease and proliferative diabetic retinopathy. While the etiology of abnormal neovascularization in diabetes is complex and paradoxical, the dysregulation of the varied processes contributing to the vascular response are due in large part to the effects of hyperglycemia. In this review, we explore the mechanisms by which hyperglycemia disrupts chemokine expression and function, including the critical hypoxia inducible factor-1 axis. We place particular emphasis on the therapeutic potential of strategies addressing these pathways; as such targeted approaches may one day help alleviate the healthcare burden of diabetic sequelae.
- Published
- 2014
18. Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs
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Dominik Duscher, Geoffrey C. Gurtner, Kevin J. Paik, Zeshaan N. Maan, Elizabeth R. Zielins, Derrick C. Wan, Alexander J. Whittam, Michael S. Hu, Ryan C. Ransom, Joseph C. Wu, Shane D. Morrison, Elizabeth A. Brett, and Michael T. Longaker
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0301 basic medicine ,Angiogenesis ,Physiology ,Molecular biology ,Cardiovascular Procedures ,lcsh:Medicine ,Pharmacology ,Cardiovascular Physiology ,Vascular Medicine ,Small hairpin RNA ,Neovascularization ,Mice ,Endocrinology ,Animal Cells ,Ischemia ,Vasculogenesis ,Medicine and Health Sciences ,Morphogenesis ,RNA, Small Interfering ,lcsh:Science ,Connective Tissue Cells ,Gene knockdown ,Multidisciplinary ,3. Good health ,Connective Tissue ,medicine.symptom ,Cellular Types ,Anatomy ,Research Article ,medicine.medical_specialty ,Endocrine Disorders ,Cell Survival ,Surgical and Invasive Medical Procedures ,Biology ,DNA construction ,Hypoxia-Inducible Factor-Proline Dioxygenases ,Diabetes Complications ,03 medical and health sciences ,Downregulation and upregulation ,Tissue Repair ,medicine ,Diabetes Mellitus ,Gene silencing ,Animals ,Wound Healing ,Revascularization ,lcsh:R ,Biology and Life Sciences ,Extremities ,Cell Biology ,Genetic Therapy ,Fibroblasts ,medicine.disease ,Hypoxia-Inducible Factor 1, alpha Subunit ,Surgery ,Research and analysis methods ,Disease Models, Animal ,030104 developmental biology ,Biological Tissue ,Molecular biology techniques ,Metabolic Disorders ,Plasmid Construction ,lcsh:Q ,Wound healing ,Physiological Processes ,Developmental Biology - Abstract
The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.
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- 2016
19. Adipose-Derived Stem Cell-Seeded Hydrogels Increase Endogenous Progenitor Cell Recruitment and Neovascularization in Wounds
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Janos A. Barrera, Zeshaan N. Maan, Robert C. Rennert, Jayakumar Rajadas, Revanth Kosaraju, Melanie Rodrigues, Geoffrey C. Gurtner, Dominik Duscher, Alexander J. Whittam, and Michael Januszyk
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0301 basic medicine ,Angiogenesis ,Cell ,Biomedical Engineering ,Neovascularization, Physiologic ,Bioengineering ,Biochemistry ,Biomaterials ,Neovascularization ,03 medical and health sciences ,Mice ,Medicine ,Animals ,Progenitor cell ,business.industry ,Mesenchymal stem cell ,Hydrogels ,Mesenchymal Stem Cells ,Original Articles ,Cell sorting ,eye diseases ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Adipose Tissue ,Self-healing hydrogels ,Wounds and Injuries ,medicine.symptom ,business ,Wound healing ,Biomedical engineering - Abstract
Adipose-derived mesenchymal stem cells (ASCs) are appealing for cell-based wound therapies because of their accessibility and ease of harvest, but their utility is limited by poor cell survival within the harsh wound microenvironment. In prior work, our laboratory has demonstrated that seeding ASCs within a soft pullulan-collagen hydrogel enhances ASC survival and improves wound healing. To more fully understand the mechanism of this therapy, we examined whether ASC-seeded hydrogels were able to modulate the recruitment and/or functionality of endogenous progenitor cells. Employing a parabiosis model and fluorescence-activated cell sorting analysis, we demonstrate that application of ASC-seeded hydrogels to wounds, when compared with injected ASCs or a noncell control, increased the recruitment of provascular circulating bone marrow-derived mesenchymal progenitor cells (BM-MPCs). BM-MPCs comprised 23.0% of recruited circulating progenitor cells in wounds treated with ASC-seeded hydrogels versus 8.4% and 2.1% in those treated with controls, p < 0.05. Exploring the potential for functional modulation of BM-MPCs, we demonstrate a statistically significant increase in BM-MPC migration, proliferation, and tubulization when exposed to hydrogel-seeded ASC-conditioned medium versus control ASC-conditioned medium (73.8% vs. 51.4% scratch assay closure; 9.1% vs. 1.4% proliferation rate; 10.2 vs. 5.5 tubules/HPF; p < 0.05 for all assays). BM-MPC expression of genes related to cell stemness and angiogenesis was also significantly increased following exposure to hydrogel-seeded ASC-conditioned medium (p < 0.05). These data suggest that ASC-seeded hydrogels improve both progenitor cell recruitment and functionality to effect greater neovascularization.
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- 2016
20. Murine Dermal Fibroblast Isolation by FACS
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Ruth Tevlin, Michael T. Longaker, Dominik Duscher, Michael S. Hu, H. Peter Lorenz, Zeshaan N. Maan, Owen Marecic, Alexander J. Whittam, Graham G. Walmsley, David Atashroo, and Geoffrey C. Gurtner
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0301 basic medicine ,Cell type ,General Chemical Engineering ,Cell ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Flow cytometry ,Dermal fibroblast ,Extracellular matrix ,Mice ,03 medical and health sciences ,medicine ,Animals ,Fibroblast ,Skin ,medicine.diagnostic_test ,General Immunology and Microbiology ,General Neuroscience ,Fibroblasts ,Flow Cytometry ,In vitro ,Extracellular Matrix ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Cell culture ,Developmental Biology - Abstract
Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin(-)) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.
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- 2016
21. Suction assisted liposuction does not impair the regenerative potential of adipose derived stem cells
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David Atashroo, Georg M. Huemer, Derrick C. Wan, Natalie Ho, Manfred Schmidt, Hans-Günther Machens, Elizabeth A. Brett, Alexander J. Whittam, Arndt F. Schilling, Graham G. Walmsley, Robert C. Rennert, Michael T. Longaker, Raphael Wenny, Zeshaan N. Maan, Michael S. Hu, Anna Luan, Dominik Duscher, Geoffrey C. Gurtner, and Michelle Lin
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0301 basic medicine ,Adult ,Male ,Pathology ,medicine.medical_specialty ,Cell Survival ,Cellular differentiation ,Population ,Adipose tissue ,Neovascularization, Physiologic ,Cell Count ,Suction ,Regenerative medicine ,General Biochemistry, Genetics and Molecular Biology ,Andrology ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Tissue engineering ,Lipectomy ,Osteogenesis ,medicine ,Animals ,Humans ,Regeneration ,Cell Lineage ,education ,Medicine(all) ,education.field_of_study ,Wound Healing ,Adipogenesis ,business.industry ,Biochemistry, Genetics and Molecular Biology(all) ,Regeneration (biology) ,Stem Cells ,Research ,Abdominoplasty ,Cell Differentiation ,General Medicine ,Stromal vascular fraction ,Middle Aged ,3. Good health ,ddc ,030104 developmental biology ,Adipose Tissue ,030220 oncology & carcinogenesis ,Female ,Stem cell ,business - Abstract
Background Adipose-derived stem cells (ASCs) have been identified as a population of multipotent cells with promising applications in tissue engineering and regenerative medicine. ASCs are abundant in fat tissue, which can be safely harvested through the minimally invasive procedure of liposuction. However, there exist a variety of different harvesting methods, with unclear impact on ASC regenerative potential. The aim of this study was thus to compare the functionality of ASCs derived from the common technique of suction-assisted lipoaspiration (SAL) versus resection. Methods Human adipose tissue was obtained from paired abdominoplasty and SAL samples from three female donors, and was processed to isolate the stromal vascular fraction. Fluorescence-activated cell sorting was used to determine ASC yield, and cell viability was assayed. Adipogenic and osteogenic differentiation capacity were assessed in vitro using phenotypic staining and quantification of gene expression. Finally, ASCs were applied in an in vivo model of tissue repair to evaluate their regenerative potential. Results SAL specimens provided significantly fewer ASCs when compared to excised fat tissue, however, with equivalent viability. SAL-derived ASCs demonstrated greater expression of the adipogenic markers FABP-4 and LPL, although this did not result in a difference in adipogenic differentiation. There were no differences detected in osteogenic differentiation capacity as measured by alkaline phosphatase, mineralization or osteogenic gene expression. Both SAL- and resection-derived ASCs enhanced significantly cutaneous healing and vascularization in vivo, with no significant difference between the two groups. Conclusion SAL provides viable ASCs with full capacity for multi-lineage differentiation and tissue regeneration, and is an effective method of obtaining ASCs for cell-based therapies.
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- 2016
22. Fibroblast-Specific Deletion of Hypoxia Inducible Factor-1 Critically Impairs Murine Cutaneous Neovascularization and Wound Healing
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Geoffrey C. Gurtner, Lauren H. Fischer, Hutton Baker, Michael Sorkin, Dominik Duscher, Graham G. Walmsley, Zeshaan N. Maan, Alexander J. Whittam, Michael S. Hu, Michael Januszyk, and Victor W. Wong
- Subjects
Male ,Hypoxia-Inducible Factor 1 ,Real-Time Polymerase Chain Reaction ,Sensitivity and Specificity ,Article ,Neovascularization ,Mice ,Random Allocation ,Ischemic insult ,Diabetes mellitus ,medicine ,Animals ,Myocardial infarction ,Fibroblast ,Cells, Cultured ,Mice, Knockout ,Wound Healing ,Neovascularization, Pathologic ,business.industry ,Anatomy ,Fibroblasts ,medicine.disease ,Hypoxia-Inducible Factor 1, alpha Subunit ,Mice, Inbred C57BL ,Disease Models, Animal ,medicine.anatomical_structure ,Gene Expression Regulation ,Cancer research ,Surgery ,medicine.symptom ,business ,Wound healing ,Gene Deletion - Abstract
Diabetes and aging are known risk factors for impaired neovascularization in response to ischemic insult, resulting in chronic wounds, and poor outcomes following myocardial infarction and cerebrovascular injury. Hypoxia-inducible factor (HIF)-1α, has been identified as a critical regulator of the response to ischemic injury and is dysfunctional in diabetic and elderly patients. To better understand the role of this master hypoxia regulator within cutaneous tissue, the authors generated and evaluated a fibroblast-specific HIF-1α knockout mouse model.The authors generated floxed HIF-1 mice (HIF-1) by introducing loxP sites around exon 1 of the HIF-1 allele in C57BL/6J mice. Fibroblast-restricted HIF-1α knockout (FbKO) mice were generated by breeding our HIF-1 with tamoxifen-inducible Col1a2-Cre mice (Col1a2-CreER). HIF-1α knockout was evaluated on a DNA, RNA, and protein level. Knockout and wild-type mice were subjected to ischemic flap and wound healing models, and CD31 immunohistochemistry was performed to assess vascularity of healed wounds.Quantitative real-time polymerase chain reaction of FbKO skin demonstrated significantly reduced Hif1 and Vegfa expression compared with wild-type. This finding was confirmed at the protein level (p0.05). HIF-1α knockout mice showed significantly impaired revascularization of ischemic tissue and wound closure and vascularity (p0.05).Loss of HIF-1α from fibroblasts results in delayed wound healing, reduced wound vascularity, and significant impairment in the ischemic neovascular response. These findings provide new insight into the importance of cell-specific responses to hypoxia during cutaneous neovascularization.
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- 2015
23. Wnt signaling induces epithelial differentiation during cutaneous wound healing
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Alexander J. Whittam, Frank Siemers, M.N. Pyles, Arash Momeni, Zeshaan N. Maan, and Khosrow Siamak Houschyar
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Embryology ,Pathology ,medicine.medical_specialty ,Cellular differentiation ,Biomedical Engineering ,Review ,Biology ,Models, Biological ,medicine ,Animals ,Humans ,Wnt Signaling Pathway ,Tissue homeostasis ,Skin ,Skin repair ,Wound Healing ,Transplantation ,integumentary system ,Regeneration (biology) ,Wnt signaling pathway ,Cell Differentiation ,Epithelial Cells ,Embryonic stem cell ,Cell biology ,Stem cell ,Corrigendum ,Wound healing ,Developmental Biology - Abstract
Cutaneous wound repair in adult mammals typically does not regenerate original dermal architecture. Skin that has undergone repair following injury is not identical to intact uninjured skin. This disparity may be caused by differences in the mechanisms that regulate postnatal cutaneous wound repair compared to embryonic skin development and thus we seek a deeper understanding of the role that Wnt signaling plays in the mechanisms of skin repair in both fetal and adult wounds. The influence of secreted Wnt signaling proteins in tissue homeostasis has galvanized efforts to identify small molecules that target Wnt-mediated cellular responses. Wnt signaling is activated by wounding and participates in every subsequent stage of the healing process from the control of inflammation and programmed cell death, to the mobilization of stem cell reservoirs within the wound site. Endogenous Wnt signaling augmentation represents an attractive option to aid in the restoration of cutaneous wounds, as the complex mechanisms of the Wnt pathway have been increasingly investigated over the years. In this review, we summarize recent data elucidating the roles that Wnt signaling plays in cutaneous wound healing process.
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- 2015
24. Exercise induces stromal cell-derived factor-1α-mediated release of endothelial progenitor cells with increased vasculogenic function
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Geoffrey C. Gurtner, Sarah Bishop, Dominik Duscher, Melanie Rodrigues, Edwin Chang, Jerry S. Chen, Arnetha J. Whitmore, Josemaria Paterno, Michael Januszyk, Alexander J. Whittam, Michael T. Longaker, Zeshaan N. Maan, and Robert C. Rennert
- Subjects
Adult ,DNA Replication ,Male ,Stromal cell ,Time Factors ,Cellular differentiation ,Paracrine Communication ,Real-Time Polymerase Chain Reaction ,Article ,Flow cytometry ,Running ,Colony-Forming Units Assay ,Vasculogenesis ,medicine ,Humans ,Progenitor cell ,Erythropoietin ,Exercise ,Endothelial Progenitor Cells ,medicine.diagnostic_test ,business.industry ,Reverse Transcriptase Polymerase Chain Reaction ,Chemotaxis ,Cell Differentiation ,Flow Cytometry ,Hypoxia-Inducible Factor 1, alpha Subunit ,Cell Hypoxia ,Chemokine CXCL12 ,Cell biology ,Blood Cell Count ,medicine.anatomical_structure ,Immunology ,Cytokines ,Surgery ,business ,Blood vessel - Abstract
Endothelial progenitor cells have been shown to traffic to and incorporate into ischemic tissues, where they participate in new blood vessel formation, a process termed vasculogenesis. Previous investigation has demonstrated that endothelial progenitor cells appear to mobilize from bone marrow to the peripheral circulation after exercise. In this study, the authors investigate potential etiologic factors driving this mobilization and investigate whether the mobilized endothelial progenitor cells are the same as those present at baseline.Healthy volunteers (n = 5) performed a monitored 30-minute run to maintain a heart rate greater than 140 beats/min. Venous blood samples were collected before, 10 minutes after, and 24 hours after exercise. Endothelial progenitor cells were isolated and evaluated.Plasma levels of stromal cell-derived factor-1α significantly increased nearly two-fold immediately after exercise, with a nearly four-fold increase in circulating endothelial progenitor cells 24 hours later. The endothelial progenitor cells isolated following exercise demonstrated increased colony formation, proliferation, differentiation, and secretion of angiogenic cytokines. Postexercise endothelial progenitor cells also exhibited a more robust response to hypoxic stimulation.Exercise appears to mobilize endothelial progenitor cells and augment their function by means of stromal cell-derived factor 1α-dependent signaling. The population of endothelial progenitor cells mobilized following exercise is primed for vasculogenesis with increased capacity for proliferation, differentiation, secretion of cytokines, and responsiveness to hypoxia. Given the evidence demonstrating positive regenerative effects of exercise, this may be one possible mechanism for its benefits.
- Published
- 2015
25. Transdermal deferoxamine prevents pressure-induced diabetic ulcers
- Author
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Mohammed Inayathullah, Dominik Duscher, Jayakumar Rajadas, Geoffrey C. Gurtner, Victor W. Wong, Melanie Rodrigues, Alexander J. Whittam, Robert C. Rennert, Michael G. Galvez, Michael Brownlee, Graham G. Walmsley, Evgenios Neofytou, Zeshaan N. Maan, Michael Januszyk, Arnetha J. Whitmore, and Andrey V. Malkovskiy
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Vascular Endothelial Growth Factor A ,Necrosis ,Neovascularization, Physiologic ,Apoptosis ,Pharmacology ,Deferoxamine ,Administration, Cutaneous ,Diabetes Mellitus, Experimental ,Neovascularization ,Diabetes Complications ,Drug Delivery Systems ,Stress, Physiological ,Diabetes mellitus ,medicine ,Pressure ,Animals ,Ulcer ,Transdermal ,Wound Healing ,Multidisciplinary ,business.industry ,Dermis ,Hypoxia (medical) ,Biological Sciences ,medicine.disease ,Mice, Inbred C57BL ,Immunology ,Drug delivery ,medicine.symptom ,business ,Wound healing ,Reactive Oxygen Species ,medicine.drug - Abstract
There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation.
- Published
- 2014
26. Aging disrupts cell subpopulation dynamics and diminishes the function of mesenchymal stem cells
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Dominik Duscher, Marcelina G. Perez, David Atashroo, Graham G. Walmsley, Ersilia Anghel, Revanth Kosaraju, Sacha M.L. Khong, Robert C. Rennert, Michael S. Hu, Michael Januszyk, Alexander J. Whittam, Zeshaan N. Maan, Atul J. Butte, and Geoffrey C. Gurtner
- Subjects
Male ,Aging ,Population ,Cell ,Adipose tissue ,Biology ,Mesenchymal Stem Cell Transplantation ,Article ,Transcriptome ,Neovascularization ,Mice ,Tubulin ,medicine ,Human Umbilical Vein Endothelial Cells ,Animals ,Cluster Analysis ,Humans ,Gene Regulatory Networks ,Progenitor cell ,education ,Cells, Cultured ,education.field_of_study ,Wound Healing ,Multidisciplinary ,Mesenchymal stem cell ,Mesenchymal Stem Cells ,Coculture Techniques ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Adipose Tissue ,Immunology ,Cancer research ,Cytokines ,medicine.symptom ,Wound healing ,Signal Transduction - Abstract
Advanced age is associated with an increased risk of vascular morbidity, attributable in part to impairments in new blood vessel formation. Mesenchymal stem cells (MSCs) have previously been shown to play an important role in neovascularization and deficiencies in these cells have been described in aged patients. Here we utilize single cell transcriptional analysis to determine the effect of aging on MSC population dynamics. We identify an age-related depletion of a subpopulation of MSCs characterized by a pro-vascular transcriptional profile. Supporting this finding, we demonstrate that aged MSCs are also significantly compromised in their ability to support vascular network formation in vitro and in vivo. Finally, aged MSCs are unable to rescue age-associated impairments in cutaneous wound healing. Taken together, these data suggest that age-related changes in MSC population dynamics result in impaired therapeutic potential of aged progenitor cells. These findings have critical implications for therapeutic cell source decisions (autologous versus allogeneic) and indicate the necessity of strategies to improve functionality of aged MSCs.
- Published
- 2014
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27. Abstract 76
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Kristine C. Rustad, Kun Ma, Peter A. Than, Geoffrey C. Gurtner, Sacha Ml Khong, Wei Liu, Marc L. Melcher, Christopher R. Davis, Paul A. Mittermiller, Michael Findlay, and Alexander J. Whittam
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business.industry ,Foundation (engineering) ,Medicine ,Surgery ,Anatomy ,Hepatic tissue ,business ,PSRC 2017 Abstract Supplement - Published
- 2017
28. Mechanotransduction and fibrosis
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Michael T. Longaker, Michael Januszyk, Victor W. Wong, Dominik Duscher, Zeshaan N. Maan, Geoffrey C. Gurtner, Alexander J. Whittam, Michael S. Hu, Arnetha J. Whitmore, Melanie Rodrigues, and Robert C. Rennert
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Pathology ,medicine.medical_specialty ,Rehabilitation ,Biomedical Engineering ,Biophysics ,Biology ,medicine.disease ,Fibrosis ,Mechanotransduction, Cellular ,Article ,Cicatrix ,Tissue fibrosis ,medicine ,Animals ,Humans ,Orthopedics and Sports Medicine ,Mechanotransduction ,Neuroscience - Abstract
Scarring and tissue fibrosis represent a significant source of morbidity in the United States. Despite considerable research focused on elucidating the mechanisms underlying cutaneous scar formation, effective clinical therapies are still in the early stages of development. A thorough understanding of the various signaling pathways involved is essential to formulate strategies to combat fibrosis and scarring. While initial efforts focused primarily on the biochemical mechanisms involved in scar formation, more recent research has revealed a central role for mechanical forces in modulating these pathways. Mechanotransduction, which refers to the mechanisms by which mechanical forces are converted to biochemical stimuli, has been closely linked to inflammation and fibrosis and is believed to play a critical role in scarring. This review provides an overview of our current understanding of the mechanisms underlying scar formation, with an emphasis on the relationship between mechanotransduction pathways and their therapeutic implications.
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- 2014
29. Abstract P18
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Zeshaan N. Maan, David Atashroo, Geoffrey C. Gurtner, Kshemendra Seanarath-Yapa, Robert C. Rennert, Michael Januszyk, Owen Marecic, Michael S. Hu, Alexander J. Whittam, Elizabeth R. Zielins, D. Duscher, Michael T. Longaker, Graham G. Walmsley, Hermann P. Lorenz, Irving L. Weissman, Kipp Weiskopf, and Ruth Tevlin
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Transplantation ,medicine.medical_specialty ,medicine.anatomical_structure ,business.industry ,Monocyte ,medicine ,Surgery ,Cutaneous wound ,business ,Dermatology - Published
- 2015
30. Abstract 9
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Dominik Duscher, Janos A. Barrera, Revanth Kosaraju, Melanie Rodrigues, Michael Januszyk, Zeshaan N. Maan, Jayakumar Rajadas, Geoffrey C. Gurtner, Alexander J. Whittam, and Robert C. Rennert
- Subjects
chemistry.chemical_compound ,chemistry ,business.industry ,Self-healing hydrogels ,Mesenchymal stem cell ,Medicine ,Adipose tissue ,Surgery ,Pullulan ,Progenitor cell ,business ,Cell biology - Published
- 2015
31. Adipose Derived Stromal Cells Obtained by Ultrasound Assisted Liposuction Versus Suction Assisted Liposuction
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Dominik Duscher, Christopher Duldulao, Derrick C. Wan, Michael T. Longaker, Ruth Tevlin, Michael S. Hu, Geoffrey C. Gurtner, Michelle Lin, Elizabeth R. Zielins, Kevin J. Paik, David Atashroo, Adrian McArdle, Alexander J. Whittam, Zeshaan N. Maan, and Graham G. Walmsley
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Suction (medicine) ,medicine.medical_specialty ,Pathology ,Stromal cell ,business.industry ,Liposuction ,medicine.medical_treatment ,medicine ,Adipose tissue ,Surgery ,business ,Ultrasound assisted - Published
- 2014
32. Melanoma Progression Depends on CXCL12 Expression by Host Endothelium
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Michael S. Hu, Arnetha J. Whitmore, Michael Januszyk, Michael T. Longaker, Alexander J. Whittam, Geoffrey C. Gurtner, Zeshaan N. Maan, Graham G. Walmsley, Dominik Duscher, and Lauren H. Fischer
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business.industry ,Medicine ,Surgery ,business ,Humanities - Abstract
Long-Term Clinical and Holistic Outcomes in Children with Cleft Lip and/or Palate: A Multidisciplinary, MixedMethods Approach Alexander C Allori, MD, MPH, Irene J Pien, Danielle L Sobol, Anna R Carlson, MD, Stephanie Watkins, MD, MSPH, Arthur S Aylsworth, MD, Robert E Meyer, PhD, Luiz Pimenta, DDS, PhD, Ronald Strauss, DMD, PhD, Jeffrey R Marcus, MD, FACS Duke University, Durham, NC, University of North Carolina, Chapel Hill, NC, Department of Health and Human Services, Raleigh, NC
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- 2015
33. Transdermal Drug Delivery of Deferoxamine Accelerates Healing and Improves Quality of Diabetic Wounds
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Zeshaan N. Maan, Graham G. Walmsley, Christopher R. Davis, Geoffrey C. Gurtner, Melanie Rodrigues, Arnetha J. Whitmore, Alexander J. Whittam, Jayakumar Rajadas, Dominik Duscher, and Michael S. Hu
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Deferoxamine ,business.industry ,Anesthesia ,media_common.quotation_subject ,Medicine ,Surgery ,Quality (business) ,business ,medicine.drug ,Transdermal ,media_common - Published
- 2014
34. Abstract 147
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Yixiao Dong, Zeshaan N. Maan, Alexander J. Whittam, Janos A. Barrera, Geoffrey C. Gurtner, Mohammed Inayatullah, Dominik Duscher, Arnetha J. Whitmore, and Jayakumar Rajadas
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Deferoxamine ,medicine.medical_specialty ,business.industry ,Anesthesia ,medicine ,Surgery ,business ,medicine.drug ,Transdermal - Published
- 2015
35. Abstract P34
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Michael T. Longaker, Lauren H. Fischer, Michael S. Hu, Geoffrey C. Gurtner, Zeshaan N. Maan, Graham G. Walmsley, Arnetha J. Whitmore, Alexander J. Whittam, and Dominik Duscher
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Endothelial stem cell ,Host (biology) ,business.industry ,Melanoma ,Cancer research ,medicine ,Surgery ,medicine.disease ,Tumor stroma ,business - Published
- 2015
36. Abstract 21
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Peter H. Lorenz, Geoffrey C. Gurtner, Michael T. Longaker, Ruth Tevlin, Elizabeth R. Zielins, Michael S. Hu, Graham G. Walmsley, Yuval Rinkevich, Zeshaan N. Maan, Irving L. Weissman, David Atashroo, Alexander J. Whittam, Michael Januszyk, and D. Duscher
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medicine.anatomical_structure ,Lineage (genetic) ,business.industry ,Medicine ,Surgery ,business ,Fibroblast ,Embryonic stem cell ,Cell biology - Published
- 2015
37. Abstract 19
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Graham G. Walmsley, Alexander J. Whittam, Zeshaan N. Maan, Michael T. Longaker, Michael S. Hu, Geoffrey C. Gurtner, Kevin J. Paik, Robert C. Rennert, Janos A. Barrera, and Dominik Duscher
- Subjects
medicine.medical_specialty ,Endocrinology ,business.industry ,Internal medicine ,medicine ,Surgery ,Type 2 diabetes ,medicine.disease ,business ,Pre adipocytes - Published
- 2015
38. Abstract 32
- Author
-
Dominik Duscher, Robert C. Rennert, Michael Januszyk, Zeshaan N. Maan, Alexander J. Whittam, Graham G. Walmsley, Michael S. Hu, and Geoffrey C. Gurtner
- Subjects
Surgery - Published
- 2015
39. Abstract 29
- Author
-
Michael T. Longaker, Zeshaan N. Maan, Ivan N. Vial, Lauren H. Fischer, Graham G. Walmsley, Alexander J. Whittam, Robert C. Rennert, Melanie Rodrigues, Geoffrey C. Gurtner, Michael Januszyk, Michael S. Hu, and Dominik Duscher
- Subjects
Circulating Progenitor Cell ,Pathology ,medicine.medical_specialty ,business.industry ,Medicine ,Surgery ,business - Published
- 2015
40. Abstract 140
- Author
-
Georg M. Huemer, Zeshaan N. Maan, Alexander J. Whittam, Anna Luan, Michael S. Hu, Dominik Duscher, Derrick C. Wan, Graham G. Walmsley, Geoffrey C. Gurtner, Michelle Lin, Manfred Schmidt, Kevin J. Paik, Michael T. Longaker, David Atashroo, and Elizabeth R. Zielins
- Subjects
medicine.medical_specialty ,business.industry ,Liposuction ,medicine.medical_treatment ,medicine ,Surgery ,Ultrasound assisted ,business - Published
- 2015
41. Endothelial Cell Derived Stromal-Derived Factor-1 (SDF-1) Regulates Neovascularization and Fibroblast Physiology in Response to Ischemia
- Author
-
Alexander J. Whittam, Arnetha J. Whitmore, Michael S. Hu, Lauren H. Fischer, Graham G. Walmsley, Geoffrey C. Gurtner, Michael T. Longaker, Zeshaan N. Maan, Robert C. Rennert, and Dominik Duscher
- Subjects
Neovascularization ,Endothelial stem cell ,Stromal cell ,medicine.anatomical_structure ,business.industry ,Cancer research ,medicine ,Ischemia ,Surgery ,medicine.symptom ,medicine.disease ,business ,Fibroblast - Published
- 2014
42. Reduced Regenerative Capacity of Aged Adipose Derived Stem Cells is Caused by Alterations of Cell Subpopulation Dynamics
- Author
-
Geoffrey C. Gurtner, Graham G. Walmsley, Michael T. Longaker, Zeshaan N. Maan, Robert C. Rennert, Dominik Duscher, Michael Januszyk, Alexander J. Whittam, David Atashroo, and Michael S. Hu
- Subjects
medicine.anatomical_structure ,business.industry ,Dynamics (mechanics) ,Cell ,Medicine ,Adipose tissue ,Surgery ,business ,Cell biology - Published
- 2014
43. LOP23
- Author
-
Geoffrey C. Gurtner, David Atashroo, Kshemendra Senarath-Yapa, Zeshaan N. Maan, M Hu, R Rennert, Ruth Tevlin, Elizabeth R. Zielins, D. Duscher, Melanie Rodrigues, Adrian McArdle, M.T. Longaker, G G Walmsley, Michael Januszyk, and Alexander J. Whittam
- Subjects
business.industry ,Adipose tissue ,Medicine ,Cell subpopulations ,Surgery ,business ,Cell biology - Published
- 2014
44. Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs.
- Author
-
Kevin J Paik, Zeshaan N Maan, Elizabeth R Zielins, Dominik Duscher, Alexander J Whittam, Shane D Morrison, Elizabeth A Brett, Ryan C Ransom, Michael S Hu, Joseph C Wu, Geoffrey C Gurtner, Michael T Longaker, and Derrick C Wan
- Subjects
Medicine ,Science - Abstract
The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.
- Published
- 2016
- Full Text
- View/download PDF
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