The loss of 5-hydroxymethylcytosine (5hmC) has been identified as a novel epigenetic hallmark for melanoma. One of the known mechanisms underlying the loss of 5hmC is the decrease in expression of ten-eleven translocation family dioxygenase (TET) genes, which encode enzymes that catalyze the generation of 5hmC. Overexpressing TET2 was shown to partially reestablish a normal 5hmC profile in melanoma and decrease invasiveness in rodents. However, the feasibility to overexpress TETs in patients remains unclear. We and others have recently demonstrated that TETs require vitamin C as a cofactor to generate 5hmC. This finding prompted us to test whether vitamin C, as an alternative to overexpressing TETs, could rebuild 5hmC content in melanoma. Consistent with previous reports, we found that the expression of TETs was decreased in various melanoma cell lines. In contrast, the expressions of sodium-dependent vitamin C transporters (SVCTs) were down-regulated in cell lines derived from melanoma metastases. Treatment of vitamin C at the physiological level (0.1 mM) promoted the content of 5hmC in melanoma cell lines derived from different stages toward the level of healthy melanocytes, which was comparable to the effect of overexpressing TET2. Vitamin C treatment decreased the malignancy of metastatic A2058 cells by inhibiting migration and anchorage-independent growth, while not exerting any effect on the rate of proliferation. Further, vitamin C treatment caused alterations in genome-wide transcriptions shown by RNA-seq, predominantly in ArhGAP30 and genes involved in extracellular matrix remodeling, which could underlie the decreased malignant phenotypes. Our data support the idea that vitamin C treatment increases 5hmC content in melanoma cells, while causing a decrease in tumor-cell invasiveness and clonogenic growth in soft agar. Thus, vitamin C could be a potential epigenetic treatment for melanoma.

Fibroblasts are stromal cells ubiquitously distributed in the body of nearly every organ tissue. These cells were previously considered to be “passive cells”, solely responsible for ensuring the turnover of the extracellular matrix (ECM). However, their versatility, including their ability to switch phenotypes in response to tissue injury and dynamic activity in the maintenance of tissue specific homeostasis and integrity have been recently revealed by the innovation of technological tools such as genetically modified mouse models and single cell analysis. These highly plastic and heterogeneous cells equipped with multifaceted functions including the regulation of angiogenesis, inflammation as well as their innate stemness characteristics, play a central role in the delicately regulated process of wound healing. Fibroblast dysregulation underlies many chronic conditions, including cardiovascular diseases, cancer, inflammatory diseases, and diabetes mellitus (DM), which represent the current major causes of morbidity and mortality worldwide. Diabetic foot ulcer (DFU), one of the most severe complications of DM affects 40 to 60 million people. Chronic non-healing DFU wounds expose patients to substantial sequelae including infections, gangrene, amputation, and death. A complete understanding of the pathophysiology of DFU and targeting pathways involved in the dysregulation of fibroblasts are required for the development of innovative new therapeutic treatments, critically needed for these patients.

Cancer stem cells (CSCs) play critical roles in cancer initiation, metastasis, recurrence and drug resistance. Recent studies have revealed involvement of cancer‐associated fibroblasts (CAF) in regulating CSCs. However, the intracellular molecular mechanisms that determine the regulatory role of CAF in modulating the plasticity of CSCs remain unknown. Here, we uncovered that intracellular Notch1 signaling in CAF serves as a molecular switch, which modulates tumor heterogeneity and aggressiveness by inversely controlling stromal regulation of the plasticity and stemness of CSCs. Using mesenchymal stem cell‐derived fibroblasts (MSC‐DF) harboring reciprocal loss‐ and gain‐of‐function Notch1 signaling, we found that MSC‐DF^{Notch1−/−} prompted co‐cultured melanoma cells to form more spheroids and acquire the phenotype (CD271⁺ and Nestin⁺) of melanoma stem/initiating cells (MIC), whereas MSC‐DF^N1IC+/+ suppressed melanoma cell sphere formation and mitigated properties of MIC. MSC‐DF^{Notch1−/−} increased stemness of CD271⁺ MIC, which resultantly exhibited stronger aggressiveness in vitro and in vivo, by up‐regulating Sox2/Oct4/Nanog expression. Consistently, when co‐grafted with melanoma cells into NSG mice, MSC‐DF^{Notch1−/−} increased, yet MSC‐DF^N1IC+/+ decreased, the amounts of CD271⁺ MIC in melanoma tissue. The amounts of CD271⁺ MIC regulated by MSC‐DF carrying high or low Notch1 pathway activity is well correlated with capability of melanoma metastasis, supporting that melanoma metastasis is MIC‐mediated. Our data demonstrate that intracellular Notch1 signaling in CAF is a molecular switch dictating the plasticity and stemness of MIC, thereby regulating melanoma aggressiveness, and therefore that targeting the intracellular Notch1 signaling pathway in CAF may present a new therapeutic strategy for melanoma. Significance Statement Tumor‐stroma interactions play an important role in cancer progression. However, the intracellular molecular mechanisms that determine the regulatory role of CAF in modulating the plasticity and stemness of CSCs remain unknown. We uncovered that intracellular Notch1 signaling in CAF is a molecular switch, inversely controlling stromal regulation of the plasticity and stemness of MIC and thereby modulating melanoma heterogeneity and aggressiveness. Our study indicates that therapeutic activation of the Notch1 pathway in CAF can control MIC plasticity and inhibit MIC‐mediated melanoma aggressiveness/metastasis. Hence, targeting the intracellular Notch1 signaling pathway in CAF may present a new therapeutic strategy for melanoma. © AlphaMed Press 2019

Progressive peripheral arterial disease (PAD) can result in chronic limb-threatening ischemia (CLTI) characterized by clinical complications including rest pain, gangrene and tissue loss. These complications can propagate even more precipitously in the setting of common concomitant diseases in patients with CLTI such as diabetes mellitus (DM). CLTI ulcers are cutaneous, non-healing wounds that persist due to the reduced perfusion and dysfunctional neovascularization associated with severe PAD. Existing therapies for CLTI are primarily limited to anatomic revascularization and medical management of contributing factors such as atherosclerosis and glycemic control. However, many patients fail these treatment strategies and are considered “no-option,” thereby requiring extremity amputation, particularly if non-healing wounds become infected or fulminant gangrene develops. Given the high economic burden imposed on patients, decreased quality of life, and poor survival of no-option CLTI patients, regenerative therapies aimed at neovascularization to improve wound healing and limb salvage hold significant promise. Cell-based therapy, specifically utilizing mesenchymal stem/stromal cells (MSCs), is one such regenerative strategy to stimulate therapeutic angiogenesis and tissue regeneration. Although previous reviews have focused primarily on revascularization outcomes after MSC treatments of CLTI with less attention given to their effects on wound healing, here we review advances in pre-clinical and clinical studies related to specific effects of MSC-based therapeutics upon ischemic non-healing wounds associated with CLTI.

Stem cell therapy has emerged as a promising approach for treatment of a number of diseases, including delayed and non-healing wounds. However, targeted systemic delivery of therapeutic cells to the dysfunctional tissues remains one formidable challenge. Herein, we present a targeted nanocarrier-mediated cell delivery method by coating the surface of the cell to be delivered with dendrimer nanocarriers modified with adhesion molecules. Infused nanocarrier-coated cells reach to destination via recognition and association with the counterpart adhesion molecules highly or selectively expressed on the activated endothelium in diseased tissues. Once anchored on the activated endothelium, nanocarriers-coated transporting cells undergo transendothelial migration, extravasation and homing to the targeted tissues to execute their therapeutic role. We now demonstrate feasibility, efficacy and safety of our targeted nanocarrier for delivery of bone marrow cells (BMC) to cutaneous wound tissues and grafted corneas and its advantages over conventional BMC transplantation in mouse models for wound healing and neovascularization. This versatile platform is suited for targeted systemic delivery of virtually any type of therapeutic cell.