All Publications


  • A Novel Xenograft Model Demonstrates Human Fibroblast Behavior During Skin Wound Repair and Fibrosis. Advances in wound care Borrelli, M., Shen, A. H., Griffin, M., Mascharak, S., Adem, S., Diaz Deleon, N. M., Ngaage, L. M., Longaker, M. T., Wan, D. C., Lorenz, H. P. 2021

    Abstract

    OBJECTIVE: Xenografts of human skin on immunodeficient mice provide a means of assessing human skin physiology and its response to wounding.APPROACH: We describe a novel xenograft model using full-thickness human neonatal foreskin to examine human skin wound repair and fibroblast heterogeneity. Full-thickness 8-mm human neonatal foreskin biopsies were sutured into the dorsum of NSG (NOD.Cg-Prkdc scidIl2rgtm1Wjl/SzJ) pups as subcutaneous grafts and exposed to cutaneous grafts at the time of weaning (postnatal day 21). To model fibrosis, xenografts were wounded with 5-mm linear incisions and monitored until post-wound day (PWD) 14. To explore whether our model can be used to test the efficacy of topical therapies, wounded xenografts were injected with fibroblast growth factor-2 (FGF2) for the first four consecutive PWDs. Xenografts were harvested for analysis by histology and fluorescence-activated cell sorting (FACS).RESULTS: Xenografts successfully engrafted with evidence of mouse-human anastomoses and resembled native neonatal foreskin at the gross and microscopic level. Wounded xenografted skin scarred with human collagen and an expansion of CD26-positive human fibroblasts. Collagen scar was quantitated by neural network analysis, which revealed distinct clustering of collagen fiber networks from unwounded skin and wounded skin at PWD7 and PWD14. Collagen fiber networks within FGF2-treated wounds at PWD14 resembled those in untreated wounded xenografts at PWD7, suggesting that FGF2 treatment at time of wounding can reduce fibrosis. Innovation and Conclusion: This novel xenograft model can be used to investigate acute fibrosis, fibroblast heterogeneity, and the efficacy of antifibrotic agents during wound repair in human skin.

    View details for DOI 10.1089/wound.2020.1392

    View details for PubMedID 34521222

  • JUN promotes hypertrophic skin scarring via CD36 in preclinical in vitro and in vivo models. Science translational medicine Griffin, M. F., Borrelli, M. R., Garcia, J. T., Januszyk, M., King, M., Lerbs, T., Cui, L., Moore, A. L., Shen, A. H., Mascharak, S., Diaz Deleon, N. M., Adem, S., Taylor, W. L., desJardins-Park, H. E., Gastou, M., Patel, R. A., Duoto, B. A., Sokol, J., Wei, Y., Foster, D., Chen, K., Wan, D. C., Gurtner, G. C., Lorenz, H. P., Chang, H. Y., Wernig, G., Longaker, M. T. 2021; 13 (609): eabb3312

    Abstract

    [Figure: see text].

    View details for DOI 10.1126/scitranslmed.abb3312

    View details for PubMedID 34516825

  • Modulating cellular responses to mechanical forces to promote wound regeneration. Advances in wound care Mascharak, S., desJardins-Park, H. E., Davitt, M. F., Guardino, N. J., Gurtner, G. C., Wan, D. C., Longaker, M. T. 2021

    Abstract

    SIGNIFICANCE: Skin scarring poses a major biomedical burden for hundreds of millions of patients annually. However, this burden could be mitigated by therapies that promote wound regeneration, with full recovery of skin's normal adnexa, matrix ultrastructure, and mechanical strength. Recent Advances: The observation of wound regeneration in several mouse models suggests a retained capacity for postnatal mammalian skin to regenerate under the right conditions. Mechanical forces are a major contributor to skin fibrosis and a prime target for devices and therapeutics that could promote skin regeneration.CRITICAL ISSUES: Wound induced hair neogenesis, Acomys "spiny" mice, Murphy Roths Large (MRL) mice, and mice treated with mechanotransduction inhibitors all show various degrees of wound regeneration. Comparison of regenerating wounds in these models against scarring wounds reveals differences in ECM interactions and in mechanosensitive activation of key signaling pathways, including Wnt, Sonic hedgehog, Focal Adhesion Kinase, and Yes-associated protein. The advent of single cell "omics" technologies has deepened this understanding and revealed that regeneration may recapitulate development in certain contexts, though it is unknown whether these mechanisms are relevant to healing in tight-skinned animals such as humans.FUTURE DIRECTIONS: While early findings in mice are promising, comparison across model systems is needed to resolve conflicting mechanisms and to identify conserved master regulators of skin regeneration. There also remains a dire need for studies on mechanomodulation of wounds in large, tight-skinned animals such as red Duroc pigs, which better approximate human wound healing.

    View details for DOI 10.1089/wound.2021.0040

    View details for PubMedID 34465219

  • Decellularized Adipose Matrices can Alleviate Radiation-induced Skin Fibrosis. Advances in wound care Adem, S., Abbas, D. B., Lavin, C., Fahy, E., Griffin, M., Diaz Deleon, N. M., Borrelli, M. R., Mascharak, S., Shen, A. H., Patel, R. A., Longaker, M. T., Nazerali, R. S., Wan, D. C. 2021

    Abstract

    OBJECTIVE: Radiation therapy is commonplace for cancer treatment but often results in fibrosis and atrophy of surrounding soft tissue. Decellularized adipose matrices (DAMs) have been reported to improve these soft tissue defects through the promotion of adipogenesis. These matrices are decellularized by a combination of physical, chemical, and enzymatic methods to minimize their immunologic effects while promoting their regenerative effects. In this study, we aimed to explore the regenerative ability of a DAM (Renuva, MTF Biologics, New Jersey, USA) in radiation-induced soft tissue injury.APPROACH: Fresh human lipoaspirate or DAM was injected into the irradiated scalp of CD-1 nude mice, and volume retention was monitored radiographically over 8 weeks. Explanted grafts were histologically assessed, and overlying skin was examined histologically and biomechanically. Irradiated human skin was also evaluated from patients following fat grafting or DAM injection. However, integrating data between murine and human skin in all cohorts is limited given the genetic variability between the two species.RESULTS: Volume retention was found to be greater with fat grafts, though DAM retention was nonetheless appreciated at irradiated sites. Improvement in both mouse and human irradiated skin overlying fat and DAM grafts was observed in terms of biomechanical stiffness, dermal thickness, collagen density, collagen fiber networks, and skin vascularity.INNOVATION: This is the first demonstration of the use of DAMs for augmenting the regenerative potential of irradiated mouse and human skin.CONCLUSIONS: These findings support use of DAMs to address soft tissue atrophy following radiation therapy. Morphological characteristics of the irradiated skin can also be improved with DAM grafting.

    View details for DOI 10.1089/wound.2021.0008

    View details for PubMedID 34346243

  • Wnt-active Engrailed-1 Lineage-negative Fibroblasts Mediate Postnatal Skin Regeneration Mascharak, S., desJardins-Park, H. E., Januszyk, M., Chen, K., Davitt, M. F., Demeter, J., Henn, D., Griffin, M., Bonham, C. A., Mooney, N., Cheng, R., Jackson, P. K., Wan, D. C., Gurtner, G. C., Longaker, M. T. WILEY. 2021: A30
  • Single Cell RNA Sequencing Reveals Fibroblast Heterogeneity Across Embryonic Origins Of Skin Griffin, M., King, M., Chen, K., desJardins-Park, H., Mascharak, S., Fahy, E., Guardino, N., Lavin, C., Abbas, D., Januszyk, M., Wan, D., Longaker, M. WILEY. 2021: A11-A12
  • Novel Genetic Analysis Of MRL Mice Reveals That Complement Inhibition By Factor H Reduces Scarring desJardins-Park, H. E., Mack, K. L., Guardino, N., Griffin, M., Davitt, M. F., Mascharak, S., Wan, D. C., Fraser, H. B., Longaker, M. T. WILEY. 2021: A13
  • Adipocytes In Dermal Wounds Undergo Conversion To Pro-fibrotic Fibroblasts That Contribute To Scar Formation Guardino, N., desJardins-Park, H. E., Griffin, M., Bauer-Rowe, K. E., King, M. E., King, M. E., Mascharak, S., Longaker, M. T. WILEY. 2021: A31
  • Transgenic Inhibition Of Engrailed-1 Results In Endogenous Postnatal Skin Regeneration Mascharak, S., desJardins-Park, H. E., Davitt, M. F., Chen, K., Griffin, M., Guardino, N., Lorenz, H., Wan, D. C., Gurtner, G. C., Longaker, M. T. WILEY. 2021: A14-A15
  • Preventing Engrailed-1 activation in fibroblasts yields wound regeneration without scarring. Science (New York, N.Y.) Mascharak, S., desJardins-Park, H. E., Davitt, M. F., Griffin, M., Borrelli, M. R., Moore, A. L., Chen, K., Duoto, B., Chinta, M., Foster, D. S., Shen, A. H., Januszyk, M., Kwon, S. H., Wernig, G., Wan, D. C., Lorenz, H. P., Gurtner, G. C., Longaker, M. T. 2021; 372 (6540)

    Abstract

    Skin scarring, the end result of adult wound healing, is detrimental to tissue form and function. Engrailed-1 lineage-positive fibroblasts (EPFs) are known to function in scarring, but Engrailed-1 lineage-negative fibroblasts (ENFs) remain poorly characterized. Using cell transplantation and transgenic mouse models, we identified a dermal ENF subpopulation that gives rise to postnatally derived EPFs by activating Engrailed-1 expression during adult wound healing. By studying ENF responses to substrate mechanics, we found that mechanical tension drives Engrailed-1 activation via canonical mechanotransduction signaling. Finally, we showed that blocking mechanotransduction signaling with either verteporfin, an inhibitor of Yes-associated protein (YAP), or fibroblast-specific transgenic YAP knockout prevents Engrailed-1 activation and promotes wound regeneration by ENFs, with recovery of skin appendages, ultrastructure, and mechanical strength. This finding suggests that there are two possible outcomes to postnatal wound healing: a fibrotic response (EPF-mediated) and a regenerative response (ENF-mediated).

    View details for DOI 10.1126/science.aba2374

    View details for PubMedID 33888614

  • Engineered Matrices Enable the Culture of Human Patient-Derived Intestinal Organoids ADVANCED SCIENCE Hunt, D. R., Klett, K. C., Mascharak, S., Wang, H. Y., Gong, D., Lou, J., Li, X., Cai, P. C., Suhar, R. A., Co, J. Y., LeSavage, B. L., Foster, A. A., Guan, Y., Amieva, M. R., Peltz, G., Xia, Y., Kuo, C. J., Heilshorn, S. C. 2021
  • Engineered Matrices Enable the Culture of Human Patient-Derived Intestinal Organoids. Advanced science (Weinheim, Baden-Wurttemberg, Germany) Hunt, D. R., Klett, K. C., Mascharak, S., Wang, H., Gong, D., Lou, J., Li, X., Cai, P. C., Suhar, R. A., Co, J. Y., LeSavage, B. L., Foster, A. A., Guan, Y., Amieva, M. R., Peltz, G., Xia, Y., Kuo, C. J., Heilshorn, S. C. 2021; 8 (10): 2004705

    Abstract

    Human intestinal organoids from primary human tissues have the potential to revolutionize personalized medicine and preclinical gastrointestinal disease models. A tunable, fully defined, designer matrix, termed hyaluronan elastin-like protein (HELP) is reported, which enables the formation, differentiation, and passaging of adult primary tissue-derived, epithelial-only intestinal organoids. HELP enables the encapsulation of dissociated patient-derived cells, which then undergo proliferation and formation of enteroids, spherical structures with polarized internal lumens. After 12 rounds of passaging, enteroid growth in HELP materials is found to be statistically similar to that in animal-derived matrices. HELP materials also support the differentiation of human enteroids into mature intestinal cell subtypes. HELP matrices allow stiffness, stress relaxation rate, and integrin-ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid-matrix interactions and potential patient-specific optimization. Organoid formation in HELP materials is most robust in gels with stiffer moduli (G' ≈ 1 kPa), slower stress relaxation rate (t1/2 ≈ 18 h), and higher integrin ligand concentration (0.5 × 10-3-1 × 10-3 m RGD peptide). This material provides a promising in vitro model for further understanding intestinal development and disease in humans and a reproducible, biodegradable, minimal matrix with no animal-derived products or synthetic polyethylene glycol for potential clinical translation.

    View details for DOI 10.1002/advs.202004705

    View details for PubMedID 34026461

    View details for PubMedCentralID PMC8132048

  • Aged skeletal stem cells generate an inflammatory degenerative niche. Nature Ambrosi, T. H., Marecic, O., McArdle, A., Sinha, R., Gulati, G. S., Tong, X., Wang, Y., Steininger, H. M., Hoover, M. Y., Koepke, L. S., Murphy, M. P., Sokol, J., Seo, E. Y., Tevlin, R., Lopez, M., Brewer, R. E., Mascharak, S., Lu, L., Ajanaku, O., Conley, S. D., Seita, J., Morri, M., Neff, N. F., Sahoo, D., Yang, F., Weissman, I. L., Longaker, M. T., Chan, C. K. 2021

    Abstract

    Loss of skeletal integrity during ageing and disease is associated with an imbalance in the opposing actions of osteoblasts and osteoclasts1. Here we show that intrinsic ageing of skeletal stem cells (SSCs)2 in mice alters signalling in the bone marrow niche and skews the differentiation of bone and blood lineages, leading to fragile bones that regenerate poorly. Functionally, aged SSCs have a decreased bone- and cartilage-forming potential but produce more stromal lineages that express high levels of pro-inflammatory and pro-resorptive cytokines. Single-cell RNA-sequencing studies link the functional loss to a diminished transcriptomic diversity of SSCs in aged mice, which thereby contributes to the transformation of the bone marrow niche. Exposure to a youthful circulation through heterochronic parabiosis or systemic reconstitution with young haematopoietic stem cells did not reverse the diminished osteochondrogenic activity of aged SSCs, or improve bone mass or skeletal healing parameters in aged mice. Conversely, the aged SSC lineage promoted osteoclastic activity and myeloid skewing by haematopoietic stem and progenitor cells, suggesting that the ageing of SSCs is a driver of haematopoietic ageing. Deficient bone regeneration in aged mice could only be returned to youthful levels by applying a combinatorial treatment of BMP2 and a CSF1 antagonist locally to fractures, which reactivated aged SSCs and simultaneously ablated the inflammatory, pro-osteoclastic milieu. Our findings provide mechanistic insights into the complex, multifactorial mechanisms that underlie skeletal ageing and offer prospects for rejuvenating the aged skeletal system.

    View details for DOI 10.1038/s41586-021-03795-7

    View details for PubMedID 34381212

  • Prrx1 Fibroblasts Represent a Pro-fibrotic Lineage in the Mouse Ventral Dermis. Cell reports Leavitt, T., Hu, M. S., Borrelli, M. R., Januszyk, M., Garcia, J. T., Ransom, R. C., Mascharak, S., desJardins-Park, H. E., Litzenburger, U. M., Walmsley, G. G., Marshall, C. D., Moore, A. L., Duoto, B., Adem, S., Foster, D. S., Salhotra, A., Shen, A. H., Griffin, M., Shen, E. Z., Barnes, L. A., Zielins, E. R., Maan, Z. N., Wei, Y., Chan, C. K., Wan, D. C., Lorenz, H. P., Chang, H. Y., Gurtner, G. C., Longaker, M. T. 2020; 33 (6): 108356

    Abstract

    Fibroblast heterogeneity has been shown within the unwounded mouse dorsal dermis, with fibroblast subpopulations being identified according to anatomical location and embryonic lineage. Using lineage tracing, we demonstrate that paired related homeobox 1 (Prrx1)-expressing fibroblasts are responsible for acute and chronic fibroses in the ventral dermis. Single-cell transcriptomics further corroborated the inherent fibrotic characteristics of Prrx1 fibroblasts during wound repair. In summary, we identify and characterize a fibroblast subpopulation in the mouse ventral dermis with intrinsic scar-forming potential.

    View details for DOI 10.1016/j.celrep.2020.108356

    View details for PubMedID 33176144

  • Peripheral Motor Neuron Activity Influences over Local Sarcoma Progression Davitt, M., Foster, D., Mascharak, S., desJardins-Park, H., Norton, J., Longaker, M. T. ELSEVIER SCIENCE INC. 2020: S230–S231
  • Detection, Scoring, and Classification of Solid Organ Fibroses with Machine Learning Analysis Mascharak, S., desJardins-Park, H. E., Davitt, M., Foster, D. S., Chinta, M., Wan, D. C., Wernig, G., Longaker, M. T. ELSEVIER SCIENCE INC. 2020: S222
  • Elucidating Molecular Drivers of Wound Regeneration in MRL Mice Via Novel Transcriptomic Analyses desJardins-Park, H. E., Mack, K. L., Davitt, M. F., Griffin, M., Mascharak, S., Fraser, H. B., Longaker, M. T. ELSEVIER SCIENCE INC. 2020: S225
  • Transdermal Deferoxamine Treatment Mitigates Fibrosis in Irradiated Skin Shen, A. H., Borrelli, M. R., Deleon, N., Adem, S., Mascharak, S., Salhotra, A., Shah, H., Longaker, M. T., Gurtner, G. C., Wan, D. C. ELSEVIER SCIENCE INC. 2020: S235
  • Wounds Heal by Tissue-Resident Fibroblast Progenitors that Proliferate Polyclonally and Mechanoresponsively Foster, D. S., Chinta, M., Salhotra, A., Nguyen, A. T., Burcham, A., Mascharak, S., Januszyk, M., Gurtner, G. C., Wernig, G., Longaker, M. T. ELSEVIER SCIENCE INC. 2020: S236–S237
  • Fibroblast Heterogeneity in Wound Healing: Hurdles to Clinical Translation. Trends in molecular medicine Mascharak, S., desJardins-Park, H. E., Longaker, M. T. 2020

    Abstract

    Recent work has revealed that fibroblasts are remarkably heterogeneous cells, but the appropriate lens through which to study this variation (lineage, phenotype, and plasticity) and its relevance to human biology remain unclear. In this opinion article, we comment on recent breakthroughs in our understanding of fibroblast heterogeneity during skin wound healing, and on open questions that must be addressed to clinically translate these findings in order to minimize scarring in patients. We emphasize the need for experimental models of wound healing that better approximate human biology, as well as comparison of scarring and regenerative phenotypes to uncover master regulators of fibrosis.

    View details for DOI 10.1016/j.molmed.2020.07.008

    View details for PubMedID 32800679

  • Rewriting the Future: Promises and Limits of Germline Gene Editing in Craniofacial Surgery. The Journal of craniofacial surgery Davitt, M., Mascharak, S., desJardins-Park, H., Chinta, M., Wan, D. C., Longaker, M. T. 2020

    View details for DOI 10.1097/SCS.0000000000006602

    View details for PubMedID 32796298

  • Prophylactic treatment with transdermal deferoxamine mitigates radiation-induced skin fibrosis. Scientific reports Shen, A. H., Borrelli, M. R., Adem, S., Deleon, N. M., Patel, R. A., Mascharak, S., Yen, S. J., Sun, B. Y., Taylor, W. L., Januszyk, M., Nguyen, D. H., Momeni, A., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2020; 10 (1): 12346

    Abstract

    Radiation therapy can result in pathological fibrosis of healthy soft tissue. The iron chelator deferoxamine (DFO) has been shown to improve skin vascularization when injected into radiated tissue prior to fat grafting. Here, we evaluated whether topical DFO administration using a transdermal drug delivery system prior to and immediately following irradiation (IR) can mitigate the chronic effects of radiation damage to the skin. CD-1 nude immunodeficient mice were split into four experimental groups: (1) IR alone (IR only), (2) DFO treatment for two weeks after recovery from IR (DFO post-IR), (3) DFO prophylaxis with treatment through and post-IR (DFO ppx), or (4) no irradiation or DFO (No IR). Immediately following IR, reactive oxygen species and apoptotic markers were significantly decreased and laser doppler analysis revealed significantly improved skin perfusion in mice receiving prophylactic DFO. Six weeks following IR, mice in the DFO post-IR and DFO ppx groups had improved skin perfusion and increased vascularization. DFO-treated groups also had evidence of reduced dermal thickness and collagen fiber network organization akin to non-irradiated skin. Thus, transdermal delivery of DFO improves tissue perfusion and mitigates chronic radiation-induced skin fibrosis, highlighting a potential role for DFO in the treatment of oncological patients.

    View details for DOI 10.1038/s41598-020-69293-4

    View details for PubMedID 32704071

  • Doxycycline Reduces Scar Thickness and Improves Collagen Architecture ANNALS OF SURGERY Moore, A. L., desJardins-Park, H. E., Duoto, B. A., Mascharak, S., Murphy, M. P., Irizarry, D. M., Foster, D. S., Jones, R. E., Barnes, L. A., Marshall, C. D., Ransom, R. C., Wernig, G., Longaker, M. T. 2020; 272 (1): 183–93
  • Radiation-induced skin fibrosis is reversed by transdermal delivery of deferoxamine Borrelli, M. R., Adem, S., Diaz, N., Mascharak, S., Sen, A., Januszyk, M., Nguyen, D., Momeni, A., Gurtner, G. C., Longaker, M. T., Wan, D. C. WILEY. 2020: S51–S52
  • Stretch marks are abundant in CD26-positive human dermal fibroblasts and exhibit increased profibrotic mechanosensitive signaling Borrelli, M. R., Griffin, M., Ngaage, L. M., Mascharak, S., Lewis, N., Januszyk, M., Wan, D. C., Longaker, M. T., Lorenz, H. P. WILEY. 2020: S32
  • Elucidating the fundamental fibrotic processes driving abdominal adhesion formation. Nature communications Foster, D. S., Marshall, C. D., Gulati, G. S., Chinta, M. S., Nguyen, A. n., Salhotra, A. n., Jones, R. E., Burcham, A. n., Lerbs, T. n., Cui, L. n., King, M. E., Titan, A. L., Ransom, R. C., Manjunath, A. n., Hu, M. S., Blackshear, C. P., Mascharak, S. n., Moore, A. L., Norton, J. A., Kin, C. J., Shelton, A. A., Januszyk, M. n., Gurtner, G. C., Wernig, G. n., Longaker, M. T. 2020; 11 (1): 4061

    Abstract

    Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients.

    View details for DOI 10.1038/s41467-020-17883-1

    View details for PubMedID 32792541

  • Understanding the impact of fibroblast heterogeneity on skin fibrosis. Disease models & mechanisms Griffin, M. F., desJardins-Park, H. E., Mascharak, S. n., Borrelli, M. R., Longaker, M. T. 2020; 13 (6)

    Abstract

    Tissue fibrosis is the deposition of excessive extracellular matrix and can occur as part of the body's natural wound healing process upon injury, or as a consequence of diseases such as systemic sclerosis. Skin fibrosis contributes to significant morbidity due to the prevalence of injuries resulting from trauma and burn. Fibroblasts, the principal cells of the dermis, synthesize extracellular matrix to maintain the skin during homeostasis and also play a pivotal role in all stages of wound healing. Although it was previously believed that fibroblasts are homogeneous and mostly quiescent cells, it has become increasingly recognized that numerous fibroblast subtypes with unique functions and morphologies exist. This Review provides an overview of fibroblast heterogeneity in the mammalian dermis. We explain how fibroblast identity relates to their developmental origin, anatomical site and precise location within the skin tissue architecture in both human and mouse dermis. We discuss current evidence for the varied functionality of fibroblasts within the dermis and the relationships between fibroblast subtypes, and explain the current understanding of how fibroblast subpopulations may be controlled through transcriptional regulatory networks and paracrine communications. We consider how fibroblast heterogeneity can influence wound healing and fibrosis, and how insight into fibroblast heterogeneity could lead to novel therapeutic developments and targets for skin fibrosis. Finally, we contemplate how future studies should be shaped to implement knowledge of fibroblast heterogeneity into clinical practice in order to lessen the burden of skin fibrosis.

    View details for DOI 10.1242/dmm.044164

    View details for PubMedID 32541065

  • Tuning Macrophage Phenotype to Mitigate Skeletal Muscle Fibrosis. Journal of immunology (Baltimore, Md. : 1950) Stepien, D. M., Hwang, C. n., Marini, S. n., Pagani, C. A., Sorkin, M. n., Visser, N. D., Huber, A. K., Edwards, N. J., Loder, S. J., Vasquez, K. n., Aguilar, C. A., Kumar, R. n., Mascharak, S. n., Longaker, M. T., Li, J. n., Levi, B. n. 2020

    Abstract

    Myeloid cells are critical to the development of fibrosis following muscle injury; however, the mechanism of their role in fibrosis formation remains unclear. In this study, we demonstrate that myeloid cell-derived TGF-β1 signaling is increased in a profibrotic ischemia reperfusion and cardiotoxin muscle injury model. We found that myeloid-specific deletion of Tgfb1 abrogates the fibrotic response in this injury model and reduces fibro/adipogenic progenitor cell proliferation while simultaneously enhancing muscle regeneration, which is abrogated by adaptive transfer of normal macrophages. Similarly, a murine TGFBRII-Fc ligand trap administered after injury significantly reduced muscle fibrosis and improved muscle regeneration. This study ultimately demonstrates that infiltrating myeloid cell TGF-β1 is responsible for the development of traumatic muscle fibrosis, and its blockade offers a promising therapeutic target for preventing muscle fibrosis after ischemic injury.

    View details for DOI 10.4049/jimmunol.1900814

    View details for PubMedID 32161098

  • Fat Grafting Rescues Radiation-Induced Joint Contracture. Stem cells (Dayton, Ohio) Borrelli, M. R., Diaz Deleon, N. M., Adem, S., Patel, R. A., Mascharak, S., Shen, A. H., Irizarry, D., Nguyen, D., Momeni, A., Longaker, M. T., Wan, D. C. 2019

    Abstract

    The aim of this study was to explore the therapeutic effects of fat grafting on radiation-induced hind limb contracture. Radiation therapy (RT) is used to palliate and/or cure a range of malignancies but causes inevitable and progressive fibrosis of surrounding soft tissue. Pathological fibrosis may lead to painful contractures which limit movement and negatively impact quality of life. Fat grafting is able to reduce and/or reverse radiation-induced soft tissue fibrosis. We explored whether fat grafting could improve extensibility in irradiated and contracted hind limbs of mice. Right hind limbs of female 60-day-old CD-1 nude mice were irradiated. Chronic skin fibrosis and limb contracture developed. After 4weeks, irradiated hind limbs were then injected with (a) fat enriched with stromal vascular cells (SVCs); (b) fat only; (c) saline; or (d) nothing (n = 10/group). Limb extension was measured at baseline and every 2weeks for 12weeks. Hind limb skin then underwent histological analysis and biomechanical strength testing. Irradiation significantly reduced limb extension but was progressively rescued by fat grafting. Fat grafting also reduced skin stiffness and reversed the radiation-induced histological changes in the skin. The greatest benefits were found in mice injected with fat enriched with SVCs. Hind limb radiation induces contracture in our mouse model which can be improved with fat grafting. Enriching fat with SVCs enhances these beneficial effects. These results underscore an attractive approach to address challenging soft tissue fibrosis in patients following RT.

    View details for DOI 10.1002/stem.3115

    View details for PubMedID 31793745

  • Endogenous Breast Cancer Shows Clonal Proliferation of Cancer Associated Fibroblasts at Primary Tumor and Metastatic Sites Foster, D. S., Chinta, M., Nguyen, A. T., Salhotra, A., Ransom, R., Jones, R., Titan, A. L., Mascharak, S., Norton, J. A., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: S262
  • JUN Drives Pathologic Scarring by Activating Key Fibroproliferative Pathways in Fibroblast Subpopulations Borrelli, M. R., Garcia, J. T., Moore, A. L., Patel, R. A., Mascharak, S., Duoto, B., Cui, L., Wan, D. C., Wernig, G., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: E215–E216
  • Fibroblast Proliferation in Wound Healing Is Clonal and Focal Adhesion Kinase-Dependent Chinta, M., Foster, D., Nguyen, A. T., Salhotra, A., Ransom, R. C., Jones, R., Titan, A. L., Marshall, C., Mascharak, S., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: S223
  • Regenerative Skin Healing Through Targeted Modulation of Engrailed1-Negative Fibroblasts Mascharak, S., desJardins-Park, H. E., Moore, A. L., Borrelli, M. R., Chinta, M., Foster, D., Lorenz, H., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: S228
  • Intrinsic Chromatin State and Extrinsic Wound-Related Cues Can Coordinate to Activate Fibroblasts for Scarring desJardins-Park, H. E., Moore, A. L., Litzenburger, U., Mascharak, S., Chinta, M., Ransom, R. C., Hu, M. S., Lorenz, H. P., Chang, H. Y., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: S223–S224
  • Cancer-Associated Fibroblasts Persist but Show Decreased Fibroblast Activation Protein Expression after Neoadjuvant Chemotherapy in Human Pancreatic Ductal Adenocarcinoma Foster, D. S., Nguyen, A. T., Chinta, M., Titan, A. L., Salhotra, A., Jones, R., Mascharak, S., Norton, J., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: S257–S258
  • Tumors Co-Opt Fibroblast Wound Healing Capacity Foster, D. S., Mascharak, S., Nguyen, A. T., Chinta, M., Salhotra, A., Titan, A. L., Jones, R., da Silva, O., Norton, J. A., Longaker, M. T. ELSEVIER SCIENCE INC. 2019: S231–S232
  • The Spectrum of Scarring in Craniofacial Wound Repair FRONTIERS IN PHYSIOLOGY desJardins-Park, H. E., Mascharak, S., Chinta, M. S., Wan, D. C., Longaker, M. T. 2019; 10
  • The Spectrum of Scarring in Craniofacial Wound Repair. Frontiers in physiology desJardins-Park, H. E., Mascharak, S. n., Chinta, M. S., Wan, D. C., Longaker, M. T. 2019; 10: 322

    Abstract

    Fibrosis is intimately linked to wound healing and is one of the largest causes of wound-related morbidity. While scar formation is the normal and inevitable outcome of adult mammalian cutaneous wound healing, scarring varies widely between different anatomical sites. The spectrum of craniofacial wound healing spans a particularly diverse range of outcomes. While most craniofacial wounds heal by scarring, which can be functionally and aesthetically devastating, healing of the oral mucosa represents a rare example of nearly scarless postnatal healing in humans. In this review, we describe the typical wound healing process in both skin and the oral cavity. We present clinical correlates and current therapies and discuss the current state of research into mechanisms of scarless healing, toward the ultimate goal of achieving scarless adult skin healing.

    View details for PubMedID 30984020

  • A Clearing Technique to Enhance Endogenous Fluorophores in Skin and Soft Tissue. Scientific reports Foster, D. S., Nguyen, A. T., Chinta, M. n., Salhotra, A. n., Jones, R. E., Mascharak, S. n., Titan, A. L., Ransom, R. C., da Silva, O. L., Foley, E. n., Briger, E. n., Longaker, M. T. 2019; 9 (1): 15791

    Abstract

    Fluorescent proteins are used extensively in transgenic animal models to label and study specific cell and tissue types. Expression of these proteins can be imaged and analyzed using fluorescent and confocal microscopy. Conventional confocal microscopes cannot penetrate through tissue more than 4-6 μm thick. Tissue clearing procedures overcome this challenge by rendering thick specimens into translucent tissue. However, most tissue clearing techniques do not satisfactorily preserve expression of endogenous fluorophores. Using simple adjustments to the BABB (Benzoic Acid Benzyl Benzoate) clearing methodology, preservation of fluorophore expression can be maintained. Modified BABB tissue clearing is a reliable technique to clear skin and soft tissue specimens for the study of dermal biology, wound healing and fibrotic pathologies.

    View details for DOI 10.1038/s41598-019-50359-x

    View details for PubMedID 31673001

  • Doxycycline Reduces Scar Thickness and Improves Collagen Architecture. Annals of surgery Moore, A. L., desJardins-Park, H. E., Duoto, B. A., Mascharak, S., Murphy, M. P., Irizarry, D. M., Foster, D. S., Jones, R. E., Barnes, L. A., Marshall, C. D., Ransom, R. C., Wernig, G., Longaker, M. T. 2018

    Abstract

    OBJECTIVE: To investigate the effects of local doxycycline administration on skin scarring.BACKGROUND: Skin scarring represents a major source of morbidity for surgical patients. Doxycycline, a tetracycline antibiotic with off-target effects on the extracellular matrix, has demonstrated antifibrotic effects in multiple organs. However, doxycycline's potential effects on skin scarring have not been explored in vivo.METHODS: Female C57BL/6J mice underwent dorsal wounding following an established splinted excisional skin wounding model. Doxycycline was administered by local injection into the wound base following injury. Wounds were harvested upon complete wound closure (postoperative day 15) for histological examination and biomechanical testing of scar tissue.RESULTS: A one-time dose of 3.90 mM doxycycline (2 mg/mL) within 12 hours of injury was found to significantly reduce scar thickness by 24.8% (P < 0.0001) without compromising tensile strength. The same effect could not be achieved by oral dosing. In doxycycline-treated scar matrices, collagen I content was significantly reduced (P = 0.0317) and fibers were favorably arranged with significantly increased fiber randomness (P = 0.0115). Common culprits of altered wound healing mechanics, including angiogenesis and inflammation, were not impacted by doxycycline treatment. However, engrailed1 profibrotic fibroblasts, responsible for scar extracellular matrix deposition, were significantly reduced with doxycycline treatment (P = 0.0005).CONCLUSIONS: Due to the substantial improvement in skin scarring and well-established clinical safety profile, locally administered doxycycline represents a promising vulnerary agent. As such, we favor rapid translation to human patients as an antiscarring therapy.

    View details for PubMedID 30585822

  • Automated Quantification of Vessel Structure: A Novel Method for Analysis of Angiogenesis in Wound Healing Jardins-Park, H., Mascharak, S., Moore, A. L., Duoto, B. A., Longaker, M. T. ELSEVIER SCIENCE INC. 2018: E196
  • Reduced Scar Thickness Achieved by Topical Doxycycline Is Mediated by Specific Skin Fibroblast Populations and Not Immune Cell Infiltrate Moore, A. L., Murphy, M. P., Irizarry, D. M., Des Jardins-Park, H. E., Duoto, B. A., Mascharak, S., Foster, D. S., Jones, R., Wernig, G., Longaker, M. T. ELSEVIER SCIENCE INC. 2018: S210–S211
  • Engrailed1-Positive Fibroblasts May Modulate Transcription of the TGF-beta Pathway in the Transition from Scarless Healing to Scarring Phenotype Moore, A. L., Marshall, C. D., Des Jardins-Park, H. E., Duoto, B. A., Mascharak, S., Barnes, A., Ransom, R. C., Hu, M. S., Lorenz, H., Longaker, M. T. ELSEVIER SCIENCE INC. 2018: E221–E222
  • Mouse Model with cJUN Over-Expression Eludes to Deep Dermal Fibroblast Expansion and Immune Cell Recruitment as the Biologic Mechanism of Hypertrophic Scarring Moore, A. L., Duoto, B. A., Des Jardins-Park, H. E., Mascharak, S., Wernig, G., Longaker, M. T. ELSEVIER SCIENCE INC. 2018: S208
  • Detecting oropharyngeal carcinoma using multispectral, narrow-band imaging and machine learning. The Laryngoscope Mascharak, S., Baird, B. J., Holsinger, F. C. 2018

    Abstract

    OBJECTIVE: To determine if multispectral narrow-band imaging (mNBI) can be used for automated, quantitative detection of oropharyngeal carcinoma (OPC).STUDY DESIGN: Prospective cohort study.METHODS: Multispectral narrow-band imaging and white light endoscopy (WLE) were used to examine the lymphoepithelial tissues of the oropharynx in a preliminary cohort of 30 patients (20 with biopsy-proven OPC, 10 healthy). Low-level image features from five patients were then extracted to train naive Bayesian classifiers for healthy and malignant tissue.RESULTS: Tumors were classified by color features with 65.9% accuracy, 66.8% sensitivity, and 64.9% specificity under mNBI. In contrast, tumors were classified with 52.3% accuracy (P=0.0108), 44.8% sensitivity (P=0.0793), and 59.9% specificity (P=0.312) under WLE. Receiver operating characteristic analysis yielded areas under the curve (AUC) of 72.3% and 54.6% for classification under mNBI and WLE, respectively (P=0.00168). For classification by both color and texture features, AUC under mNBI increased (80.1%, P=0.00230) but did not improve under WLE (below 55% for both models, P=0.180). Cross-validation with five folds yielded an AUC above 80% for both mNBI models and below 55% for both WLE models (P=0.0000410 and 0.000116).CONCLUSION: Compared to WLE, mNBI significantly enhanced the performance of a naive Bayesian classifier trained on low-level image features of oropharyngeal mucosa. These findings suggest that automated clinical detection of OPC might be used to enhance surgical vision, improve early diagnosis, and allow for high-throughput screening.LEVEL OF EVIDENCE: NA. Laryngoscope, 2018.

    View details for PubMedID 29577322

  • Prrx1 Labels the Fibrogenic Fibroblast in the Ventral Dermis Hu, M., Leavitt, T., Garcia, J., Ransom, R., Litzenburger, U., Walmsley, G., Marshall, C., Moore, A., Mascharak, S., Chan, C., Wan, D., Lorenz, P., Chang, H., Longaker, M. WILEY. 2018: A4
  • YAP-dependent mechanotransduction is required for proliferation and migration on native-like substrate topography BIOMATERIALS Mascharak, S., Benitez, P. L., Proctor, A. C., Madl, C. M., Hu, K. H., Dewi, R. E., Butte, M. J., Heilshorn, S. C. 2017; 115: 155-166

    Abstract

    Native vascular extracellular matrices (vECM) consist of elastic fibers that impart varied topographical properties, yet most in vitro models designed to study the effects of topography on cell behavior are not representative of native architecture. Here, we engineer an electrospun elastin-like protein (ELP) system with independently tunable, vECM-mimetic topography and demonstrate that increasing topographical variation causes loss of endothelial cell-cell junction organization. This loss of VE-cadherin signaling and increased cytoskeletal contractility on more topographically varied ELP substrates in turn promote YAP activation and nuclear translocation, resulting in significantly increased endothelial cell migration and proliferation. Our findings identify YAP as a required signaling factor through which fibrous substrate topography influences cell behavior and highlights topography as a key design parameter for engineered biomaterials.

    View details for DOI 10.1016/j.biomaterials.2016.11.019

    View details for Web of Science ID 000390642100014

    View details for PubMedID 27889666

  • Response to Open Peer Commentaries on "Human Germline CRISPR-Cas Modification: Toward a Regulatory Framework". American journal of bioethics Evitt, N. H., Mascharak, S., Altman, R. B. 2016; 16 (10): W1-2

    View details for DOI 10.1080/15265161.2016.1214308

    View details for PubMedID 27653416

  • Use of protein-engineered fabrics to identify design rules for integrin ligand clustering in biomaterials INTEGRATIVE BIOLOGY Benitez, P. L., Mascharak, S., Proctor, A. C., Heilshorn, S. C. 2016; 8 (1): 50-61

    Abstract

    While ligand clustering is known to enhance integrin activation, this insight has been difficult to apply to the design of implantable biomaterials because the local and global ligand densities that enable clustering-enhanced integrin signaling were unpredictable. Here, two general design principles for biomaterial ligand clustering are elucidated. First, clustering ligands enhances integrin-dependent signals when the global ligand density, i.e., the ligand density across the cellular length scale, is near the ligand's effective dissociation constant (KD,eff). Second, clustering ligands enhances integrin activation when the local ligand density, i.e., the ligand density across the length scale of individual focal adhesions, is less than an overcrowding threshold. To identify these principles, we fabricated a series of elastin-like, electrospun fabrics with independent control over the local (0 to 122 000 ligands μm(-2)) and global (0 to 71 000 ligand μm(-2)) densities of an arginine-glycine-aspartate (RGD) ligand. Antibody blocking studies confirmed that human umbilical vein endothelial cell adhesion to these protein-engineered biomaterials was primarily due to αVβ3 integrin binding. Clustering ligands enhanced cell proliferation, focal adhesion number, and focal adhesion kinase expression near the ligand's KD,eff of 12 000 RGD μm(-2). Near this global ligand density, cells on ligand-clustered fabrics behaved similarly to cells grown on fabrics with significantly larger global ligand densities but without clustering. However, this enhanced ligand-clustering effect was not observed above a threshold cut-off concentration. At a local ligand density of 122 000 RGD μm(-2), cell division, focal adhesion number, and focal adhesion kinase expression were significantly reduced relative to fabrics with identical global ligand density and lesser local ligand densities. Thus, when clustering results in overcrowding of ligands, integrin receptors are no longer able to effectively engage with their target ligands. Together, these two insights into the cellular responses to ligand clustering at the cell-matrix interface may serve as design principles when developing future generations of implantable biomaterials.

    View details for DOI 10.1039/c5ib00258c

    View details for Web of Science ID 000368348900006

    View details for PubMedCentralID PMC4771524

  • Use of protein-engineered fabrics to identify design rules for integrin ligand clustering in biomaterials. Integrative biology : quantitative biosciences from nano to macro Benitez, P. L., Mascharak, S. n., Proctor, A. C., Heilshorn, S. C. 2016; 8 (1): 50–61

    Abstract

    While ligand clustering is known to enhance integrin activation, this insight has been difficult to apply to the design of implantable biomaterials because the local and global ligand densities that enable clustering-enhanced integrin signaling were unpredictable. Here, two general design principles for biomaterial ligand clustering are elucidated. First, clustering ligands enhances integrin-dependent signals when the global ligand density, i.e., the ligand density across the cellular length scale, is near the ligand's effective dissociation constant (KD,eff). Second, clustering ligands enhances integrin activation when the local ligand density, i.e., the ligand density across the length scale of individual focal adhesions, is less than an overcrowding threshold. To identify these principles, we fabricated a series of elastin-like, electrospun fabrics with independent control over the local (0 to 122 000 ligands μm(-2)) and global (0 to 71 000 ligand μm(-2)) densities of an arginine-glycine-aspartate (RGD) ligand. Antibody blocking studies confirmed that human umbilical vein endothelial cell adhesion to these protein-engineered biomaterials was primarily due to αVβ3 integrin binding. Clustering ligands enhanced cell proliferation, focal adhesion number, and focal adhesion kinase expression near the ligand's KD,eff of 12 000 RGD μm(-2). Near this global ligand density, cells on ligand-clustered fabrics behaved similarly to cells grown on fabrics with significantly larger global ligand densities but without clustering. However, this enhanced ligand-clustering effect was not observed above a threshold cut-off concentration. At a local ligand density of 122 000 RGD μm(-2), cell division, focal adhesion number, and focal adhesion kinase expression were significantly reduced relative to fabrics with identical global ligand density and lesser local ligand densities. Thus, when clustering results in overcrowding of ligands, integrin receptors are no longer able to effectively engage with their target ligands. Together, these two insights into the cellular responses to ligand clustering at the cell-matrix interface may serve as design principles when developing future generations of implantable biomaterials.

    View details for PubMedID 26692238

  • Human Germline CRISPR-Cas Modification: Toward a Regulatory Framework. American journal of bioethics Evitt, N. H., Mascharak, S., Altman, R. B. 2015; 15 (12): 25-29

    Abstract

    CRISPR germline editing therapies (CGETs) hold unprecedented potential to eradicate hereditary disorders. However, the prospect of altering the human germline has sparked a debate over the safety, efficacy, and morality of CGETs, triggering a funding moratorium by the NIH. There is an urgent need for practical paths for the evaluation of these capabilities. We propose a model regulatory framework for CGET research, clinical development, and distribution. Our model takes advantage of existing legal and regulatory institutions but adds elevated scrutiny at each stage of CGET development to accommodate the unique technical and ethical challenges posed by germline editing.

    View details for DOI 10.1080/15265161.2015.1104160

    View details for PubMedID 26632357