Fibroblasts in Diabetic Foot Ulcers.

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.

Identification of unique transcriptomic signatures through integrated multispecies comparative analysis and WGCNA in bovine oocyte development.

BACKGROUND: Cattle (Bos taurus) are a major large livestock, however, compared with other species, the transcriptional specificity of bovine oocyte development has not been emphasised. RESULTS: To reveal the unique transcriptional signatures of bovine oocyte development, we used integrated multispecies comparative analysis and weighted gene co-expression network analysis (WGCNA) to perform bioinformatic analysis of the germinal follicle (GV) and second meiosis (MII) gene expression profile from cattle, sheep, pigs and mice. We found that the expression levels of most genes were down-regulated from GV to MII in all species. Next, the multispecies comparative analysis showed more genes involved in the regulation of cAMP signalling during bovine oocyte development. Moreover, the green module identified by WGCNA was closely related to bovine oocyte development. Finally, integrated multispecies comparative analysis and WGCNA picked up 61 bovine-specific signature genes that participate in metabolic regulation and steroid hormone biosynthesis. CONCLUSION: In a short, this study provides new insights into the regulation of cattle oocyte development from a cross-species comparison.

Novel Gene-Modified Mesenchymal Stem Cell Therapy Reverses Impaired Wound Healing in Ischemic Limbs.

OBJECTIVE: Here, we report a new method to increase the therapeutic potential of mesenchymal stem/stromal cells (MSCs) for ischemic wound healing. We tested biological effects of MSCs modified with E-selectin, a cell adhesion molecule capable of inducing postnatal neovascularization, on a translational murine model. BACKGROUND: Tissue loss significantly worsens the risk of extremity amputation for patients with chronic limb-threatening ischemia. MSC-based therapeutics hold major promise for wound healing and therapeutic angiogenesis, but unmodified MSCs demonstrate only modest benefits. METHODS: Bone marrow cells harvested from FVB/ROSA26Sor mTmG donor mice were transduced with E-selectin-green fluorescent protein (GFP)/AAV-DJ or GFP/AAV-DJ (control). Ischemic wounds were created via a 4 mm punch biopsy in the ipsilateral limb after femoral artery ligation in recipient FVB mice and subsequently injected with phosphate-buffered saline or 1×10 6 donor MSC GFP or MSC E-selectin-GFP . Wound closure was monitored daily for 7 postoperative days, and tissues were harvested for molecular and histologic analysis and immunofluorescence. Whole-body DiI perfusion and confocal microscopy were utilized to evaluate wound angiogenesis. RESULTS: Unmodified MSCs do not express E-selectin, and MSC E-selectin-GFP gain stronger MSC phenotype yet maintain trilineage differentiation and colony-forming capability. MSC E-selectin-GFP therapy accelerates wound healing compared with MSC GFP and phosphate-buffered saline treatment. Engrafted MSC E-selectin-GFP manifest stronger survival and viability in wounds at postoperative day 7. Ischemic wounds treated with MSC E-selectin-GFP exhibit more abundant collagen deposition and enhanced angiogenic response. CONCLUSIONS: We establish a novel method to potentiate regenerative and proangiogenic capability of MSCs by modification with E-selectin/adeno-associated virus. This innovative therapy carries the potential as a platform worthy of future clinical studies.

Intracellular Notch1 Signaling in Cancer-Associated Fibroblasts Dictates the Plasticity and Stemness of Melanoma Stem/Initiating Cells.

Cancer stem cells (CSCs) play critical roles in cancer initiation, metastasis, recurrence, and drug resistance. Recent studies have revealed involvement of cancer-associated fibroblasts (CAFs) in regulating CSCs. However, the intracellular molecular mechanisms that determine the regulatory role of CAFs in modulating the plasticity of CSCs remain unknown. Here, we uncovered that intracellular Notch1 signaling in CAFs 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-of-function and gain-of-function Notch1 signaling, we found that MSC-DFNotch1-/- prompted cocultured melanoma cells to form more spheroids and acquire the phenotype (CD271+ and Nestin+ ) of melanoma stem/initiating cells (MICs), whereas MSC-DFN1IC+/+ suppressed melanoma cell sphere formation and mitigated properties of MICs. MSC-DFNotch1-/- increased stemness of CD271+ MIC, which resultantly exhibited stronger aggressiveness in vitro and in vivo, by upregulating Sox2/Oct4/Nanog expression. Consistently, when cografted with melanoma cells into NOD scid gamma (NSG) mice, MSC-DFNotch1-/- increased, but MSC-DFN1IC+/+ 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 CAFs is a molecular switch dictating the plasticity and stemness of MICs, thereby regulating melanoma aggressiveness, and therefore that targeting the intracellular Notch1 signaling pathway in CAFs may present a new therapeutic strategy for melanoma. Stem Cells 2019;37:865-875.

c-Kit suppresses atherosclerosis in hyperlipidemic mice.

Atherosclerosis is the most common underlying cause of cardiovascular morbidity and mortality worldwide. c-Kit (CD117) is a member of the receptor tyrosine kinase family, which regulates differentiation, proliferation, and survival of multiple cell types. Recent studies have shown that c-Kit and its ligand stem cell factor (SCF) are present in arterial endothelial cells and smooth muscle cells (SMCs). The role of c-Kit in cardiovascular disease remains unclear. The aim of the current study is to determine the role of c-Kit in atherogenesis. For this purpose, atherosclerotic plaques were quantified in c-Kit-deficient mice (KitMut) after they were fed a high-fat diet (HFD) for 16 wk. KitMut mice demonstrated substantially greater atherosclerosis compared with control (KitWT) littermates (P < 0.01). Transplantation of c-Kit-positive bone marrow cells into KitMut mice failed to rescue the atherogenic phenotype, an indication that increased atherosclerosis was associated with reduced arterial c-Kit. To investigate the mechanism, SMC organization and morphology were analyzed in the aorta by histopathology and electron microscopy. SMCs were more abundant, disorganized, and vacuolated in aortas of c-Kit mutant mice compared with controls (P < 0.05). Markers of the "contractile" SMC phenotype (calponin, SM22α) were downregulated with pharmacological and genetic c-Kit inhibition (P < 0.05). The absence of c-Kit increased lipid accumulation and significantly reduced the expression of the ATP-binding cassette transporter G1 (ABCG1) necessary for lipid efflux in SMCs. Reconstitution of c-Kit in cultured KitMut SMCs resulted in increased spindle-shaped morphology, reduced proliferation, and elevated levels of contractile markers, all indicators of their restored contractile phenotype (P < 0.05).NEW & NOTEWORTHY This study describes the novel vasculoprotective role of c-Kit against atherosclerosis and its function in the preservation of the SMC contractile phenotype.

The effect of estrogen on diabetic wound healing is mediated through increasing the function of various bone marrow-derived progenitor cells.

OBJECTIVE: Endothelial progenitor cells (EPCs) are the key cells of postnatal neovascularization, and mesenchymal stem cells (MSCs) possess pluripotent differentiation capacity and contribute to tissue regeneration and wound healing. Both EPCs and MSCs are critical to the wound repair process, which is hindered in diabetes mellitus. Diabetes has been shown to decrease the function of these progenitor cells, whereas estrogen has beneficial wound healing effects. However, the role of estrogen in modulating EPC and MSC biology in diabetes is unknown. We investigated the effect of estrogen on improving bone marrow (BM)-derived EPC and MSC function using a murine diabetic wound healing model. METHODS: Female diabetic db+/db+ and nondiabetic control mice were wounded cutaneously and treated with topical estrogen or placebo cream. On day 5 after wounding, BM cells were harvested to quantify EPC number and colony-forming units of EPCs and MSCs. Wound healing rate was concurrently studied. Vessel density and scar density were then quantified using whole body perfusion and laser confocal microscopy. EPC recruitment was documented by immunohistochemistry to identify CD34- and vascular endothelial growth factor receptor 2-positive cells in the vessel wall. Data were analyzed by analysis of variance. RESULTS: Topical estrogen significantly increased colony-forming units of both EPCs and MSCs compared with placebo treatment, indicating improved viability and proliferative ability of these cells. Consistently, increased recruitment of EPCs to diabetic wounds and higher vessel density were observed in estrogen-treated compared with placebo-treated mice. Consequently, topical estrogen significantly accelerated wound healing as early as day 6 after wounding. In addition, scar density resulting from collagen deposition was increased in the estrogen-treated group, reflecting increased MSC activity and differentiation. CONCLUSIONS: Estrogen treatment increases wound healing and wound neovascularization in diabetic mice. Our data implicate that these beneficial effects may be mediated through improving the function of BM-derived EPCs and MSCs.

Intramuscular E-selectin/adeno-associated virus gene therapy promotes wound healing in an ischemic mouse model.

BACKGROUND: Poor wound healing in critical limb ischemia (CLI) is attributed to impaired neovascularization and reperfusion. Optimizing the ischemic wound with adhesion molecules that enhance stem cell homing may revolutionize treatment. The purpose of this study is to test the efficacy of adhesion molecule E-selectin on wound healing in an ischemic mouse wound. METHODS: Adult FVB/NJ mice underwent unilateral femoral artery and vein ligation to induce CLI. A 4-mm punch biopsy wound was created on the anterior thigh to simulate ischemic wounds. Intramuscular injection of adeno-associated virus (AAV) carrying either E-selectin (E-selectin/AAV, n = 11) or LacZ as control (LacZ/AAV, n = 10) was performed. Gross wound size was measured for 10 d postoperatively. Ischemic hindlimb reperfusion was quantified using laser Doppler imaging. Wound tissue neovascularization was visualized using DiI perfusion and confocal microscopy. E-selectin expression in wounds was verified by immunofluorescence. RESULTS: Immunofluorescence confirmed E-selectin/AAV delivery in treatment versus control limbs. Wounds from E-selectin/AAV mice versus controls revealed surface area healing of 54% versus 20% (P < 0.01) on postoperative day (POD) 1, 78% versus 51% on POD 4 (P < 0.01), and 97% versus 84% on POD 10 (P < 0.01). Laser Doppler imaging revealed greater reperfusion in E-selectin/AAV mice versus controls by POD 10 (0.49 versus 0.27, P < 0.05). DiI perfused ligated hindlimb in E-selectin/AAV versus control mice revealed mean neovascularization intensity score of 30 versus 18 (P < 0.05) on POD 10. CONCLUSIONS: Intramuscularly injected E-selectin/AAV gene therapy in mice with CLI significantly increases wound angiogenesis and limb reperfusion, expediting overall wound healing.

A Reliable Mouse Model of Hind limb Gangrene.

BACKGROUND: Lack of a reliable hind limb gangrene animal model limits preclinical studies of gangrene, a severe form of critical limb ischemia. We develop a novel mouse hind limb gangrene model to facilitate translational studies. METHODS: BALB/c, FVB, and C57BL/6 mice underwent femoral artery ligation (FAL) with or without administration of NG-nitro-L-arginine methyl ester (L-NAME), an endothelial nitric oxide synthase inhibitor. Gangrene was assessed using standardized ischemia scores ranging from 0 (no gangrene) to 12 (forefoot gangrene). Laser Doppler imaging (LDI) and DiI perfusion quantified hind limb reperfusion postoperatively. RESULTS: BALB/c develops gangrene with FAL-only (n = 11/11, 100% gangrene incidence), showing mean limb ischemia score of 12 on postoperative days (PODs) 7 and 14 with LDI ranging from 0.08 to 0.12 on respective PODs. Most FVB did not develop gangrene with FAL-only (n = 3/9, 33% gangrene incidence) but with FAL and L-NAME (n = 9/9, 100% gangrene incidence). Mean limb ischemia scores for FVB undergoing FAL with L-NAME were significantly higher than for FVB receiving FAL-only. LDI score and capillary density by POD 28 were significantly lower in FVB undergoing FAL with L-NAME. C57BL/6 did not develop gangrene with FAL-only or FAL and L-NAME. CONCLUSIONS: Reproducible murine gangrene models may elucidate molecular mechanisms for gangrene development, facilitating therapeutic intervention.

Hyperbaric oxygen therapy activates hypoxia-inducible factor 1 (HIF-1), which contributes to improved wound healing in diabetic mice.

Hyperbaric oxygen (HBO) therapy has been used as an adjunctive therapy for diabetic foot ulcers, although its mechanism of action is not completely understood. Recently, it has been shown that HBO mobilizes the endothelial progenitor cells (EPCs) from bone marrow that eventually will aggregate in the wound. However, the gathering of the EPCs in diabetic wounds is impaired because of the decreased levels of local stromal-derived factor-1α (SDF-1α). Therefore, we investigated the influence of HBO on hypoxia-inducible factor 1 (HIF-1), which is a central regulator of SDF-1α and is down-regulated in diabetic wounds. The effects of HBO on HIF-1α function were studied in human dermal fibroblasts, SKRC7 cells, and HIF-1α knock-out and wild-type mouse embryonic fibroblasts using appropriate techniques (Western blot, quantitative polymerase chain reaction, and luciferase hypoxia-responsive element reporter assay). Cellular proliferation was assessed using H(3) -thymidine incorporation assay. The effect of HIF in combination with HBOT was tested by inoculating stable HIF-1α-expressing adenovirus (Adv-HIF) into experimental wounds in db/db mice exposed to HBO. HBO activates HIF-1α at several levels by increasing both HIF-1α stability (by a non-canonical mechanism) and activity (as shown both by induction of relevant target genes and by a specific reporter assay). HIF-1α induction has important biological relevance because the induction of fibroblast proliferation in HBO disappears when HIF-1α is knocked down. Moreover, the local transfer of stable HIF-1α-expressing adenovirus (Adv-HIF) into experimental wounds in diabetic (db/db mice) animals has an additive effect on HBO-mediated improvements in wound healing. In conclusion, HBO stabilizes and activates HIF-1, which contributes to increased cellular proliferation. In diabetic animals, the local transfer of active HIF further improves the effects of HBO on wound healing.

Characterization of interleukin-1ß as a proinflammatory cytokine in grass carp (Ctenopharyngodon idella).

Interleukin-1ß (IL-1ß) is a well-characterized cytokine that plays key roles in cellular responses to infection, inflammation, and immunological challenges in mammals. In this study, we identified and analyzed a grass carp (Ctenopharyngodon idella) ortholog of IL-1ß (gcIL-1ß), examined its expression patterns in various tissues in both healthy and lipopolysaccharide (LPS)-stimulated specimens, and evaluated its proinflammatory activities. The gcIL-1ß gene consists of seven exons and six introns. The full-length cDNA sequence contains an open reading frame of 813 nucleotides. The deduced amino acid sequence exhibits a characteristic IL-1 signature but lacks the typical IL-1ß converting enzyme cleavage site that is conserved in mammals. In the phylogenetic tree, IL-1ßs from grass carp and other members of the Cyprinidae family clustered into a single group. Expression pattern analysis revealed that gcIL-1ß is constitutively expressed in all 11 tissues examined, and LPS stimulation leads to significant up-regulation in muscle, liver, intestine, skin, trunk kidney, head kidney, and gill. Recombinant grass carp IL-1ß (rgcIL-1ß) was generated prokaryotically as a fusion protein of Trx-rgcIL-1ß. An anti-rgcIL-1ß polyclonal antibody (rgcIL-1ß pAb) was raised in mice against the purified Trx-rgcIL-1ß. Western blot analysis confirmed that rgcIL-1ß pAb reacted specifically with gcIL-1ß in C. idella kidney (CIK) cells. Quantitative real-time PCR data indicated that intestinal mRNA expression levels of endogenous IL-1ß, IL-1R2, and TNF-α were significantly up-regulated following Trx-rgcIL-1ß exposure. The inhibitory activities of rgcIL-1ß pAb against the inflammatory response were confirmed in a model of Aeromonas hydrophila-induced intestinal inflammation. Our immunohistochemical study revealed that the degree and intensity of inflammatory cell infiltration are fully consistent with the observed mRNA expression patterns of these key inflammatory genes. Taken together, these data suggest that gcIL-1ß plays a critical role in the proinflammatory response in the grass carp intestine.

Directing Cell Delivery to Murine Atherosclerotic Aortic Lesions via Targeting Inflamed Circulatory Interface using Nanocarriers.

Stem cell therapy holds significant potential for many inflammatory diseases and regenerative medicine applications. However, delivery of therapeutic cells to specific disease sites after systemic administration without indiscriminate trafficking to other non-target tissues is a major limitation of current cell therapies. Here, we describe a novel nanocarrier-directed targeted cell delivery system that enables cell surface coating with dendrimer nanocarriers containing adhesion moieties to serve as a global positioning system "GPS" to guide circulating cells to targeted lesions and mediate the anchoring of cells at the inflammation site. By exploiting cell surface ligands/receptors selectively and/or molecular moieties that are highly expressed on activated endothelium in pathologic disease states, nanocarrier-coated cells containing the counterpart binding receptors/ligands can be enabled to specifically traffic to and dock at vasculature within target lesions. We demonstrate the efficacy of the I-domain fragment of LFA-1 ( id LFA-1) complexed to modified nanocarriers to facilitate homing of mesenchymal stem cells (MSCs) to inflamed luminal endothelial cells on which ICAM-1 is highly expressed in a murine model of aortic atherosclerosis. Our method can overcome challenges imposed by the high velocity and dynamic circulatory flow of the aorta to successfully deliver MSCs to atherosclerotic regions and allow for docking of the potentially therapeutic and immunomodulating cells. This targeted cell-delivery platform can be tailored for selective systemic delivery of various types of therapeutic cells to different disease areas.

Methods and Limitations of Augmenting Mesenchymal Stem Cells for Therapeutic Applications.

Significance: Given their capacity for self-renewal, multilineage differentiation, and immunomodulatory potential, mesenchymal stem cells (MSCs) represent a promising modality of clinical therapy for both regenerative medicine and immune diseases. In this study, we review the key approaches and popular methods utilized to boost potency and modify functions of MSCs for clinical purposes as well as their associated limitations. Recent Advances: Several major domains of cell modification strategies are currently employed by investigators to overcome these deficits and augment the therapeutic potential of MSCs. Priming MSCs with soluble factors or pharmacologic agents as well as manipulating oxygen availability in culture have been demonstrated to be effective biochemical methods to augment MSC potential. Distinct genetic and epigenetic methods have emerged in recent years to modify the genetic expression of target proteins and factors thereby modulating MSCs capacity for differentiation, migration, and proliferation. Physical methods utilizing three-dimensional culture methods and alternative cell delivery systems and scaffolds can be used to recapitulate the native MSC niche and augment their engraftment and viability for in vivo models. Critical Issues: Unmodified MSCs have demonstrated only modest benefits in many preclinical and clinical studies due to issues with cell engraftment, viability, heterogeneity, and immunocompatibility between donor and recipient. Furthermore, unmodified MSCs can have low inherent therapeutic potential for which intensive research over the past few decades has been dedicated to improving cell functionality and potency.

Targeted cell delivery of mesenchymal stem cell therapy for cardiovascular disease applications: a review of preclinical advancements.

Cardiovascular diseases (CVD) continue to be the leading cause of morbidity and mortality globally and claim the lives of over 17 million people annually. Current management of CVD includes risk factor modification and preventative strategies including dietary and lifestyle changes, smoking cessation, medical management of hypertension and cholesterol lipid levels, and even surgical revascularization procedures if needed. Although these strategies have shown therapeutic efficacy in reducing major adverse cardiovascular events such as heart attack, stroke, symptoms of chronic limb-threatening ischemia (CLTI), and major limb amputation significant compliance by patients and caregivers is required and off-target effects from systemic medications can still result in organ dysfunction. Stem cell therapy holds major potential for CVD applications but is limited by the low quantities of cells that are able to traffic to and engraft at diseased tissue sites. New preclinical investigations have been undertaken to modify mesenchymal stem cells (MSCs) to achieve targeted cell delivery after systemic administration. Although previous reviews have focused broadly on the modification of MSCs for numerous local or intracoronary administration strategies, here we review recent preclinical advances related to overcoming challenges imposed by the high velocity and dynamic flow of the circulatory system to specifically deliver MSCs to ischemic cardiac and peripheral tissue sites. Many of these technologies can also be applied for the targeted delivery of other types of therapeutic cells for treating various diseases.

Mesenchymal stem cell-based therapy for non-healing wounds due to chronic limb-threatening ischemia: A review of preclinical and clinical studies.

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.

Racial and ethnic minorities in lower income brackets are associated with late-stage diagnosis of non-ocular melanoma.

INTRODUCTION: Despite advances in oncologic care, racial and socio-economic outcome disparities persist in non-ocular melanoma patients. However, the unmet need is understanding the population at risk for late tumor stage at diagnosis. We sought to analyze the groups with an increased risk of unfavorable tumor stage at diagnosis. METHODS: Patients with non-ocular melanoma were reviewed using the 2000-2019 SEER Research Data (SEER*Stat) and grouped into early tumor stage at diagnosis (stage I-IIC) and late (stage III-IVC). Multivariable logistic and Cox regression examined the association of demographic, socioeconomic, and clinical factors with late-stage diagnosis and overall survival, respectively. Kaplan-Meier estimates were calculated with racial and county-level household income stratification to evaluate overall survival differences. RESULTS: Of 147,606 patients diagnosed with non-ocular melanoma, 38,695 cases were identified based on inclusion and exclusion criteria and separated into those with early-stage diagnosis (median 63 years) and those with late-stage (median 62 years). Male gender, Black race, Asian or Pacific Islander race, and Hispanic ethnicity were significantly associated with late-stage tumor diagnosis (p < 0.001). Receipt of surgery and a median county-level household income >$75,000 were protective for late-stage tumor diagnosis (p < 0.001). Additionally, male gender, Black, Asian or Pacific Islander, American Indian/Alaskan Native races, metastasis, and late-stage diagnosis were associated with factors significantly associated with decreased overall survival (p-value <0.001). Receipt of surgery and a median household income of $50,000-$74,999 and >$75,000 were factors associated with increased overall survival (p < 0.001). The median overall survival was 89 months, but Black patients (58 months) and <$50,000 income households (75 months) had significantly worse survival (p < 0.001). CONCLUSIONS: Hispanic ethnicity, Black and Asian or Pacific Islander race, and low-income households were associated with late-stage non-ocular melanoma at diagnosis. Black, Asian or Pacific Islander and American Indian/Alaskan Native races and lower-income households were associated with worse overall survival. Identifying addressable causal factors that link this at-risk population to poor cancer prognosis is warranted.

E-Selectin/AAV Gene Therapy Promotes Myogenesis and Skeletal Muscle Recovery in a Mouse Hindlimb Ischemia Model.

The response to ischemia in peripheral artery disease (PAD) depends on compensatory neovascularization and coordination of tissue regeneration. Identifying novel mechanisms regulating these processes is critical to the development of nonsurgical treatments for PAD. E-selectin is an adhesion molecule that mediates cell recruitment during neovascularization. Therapeutic priming of ischemic limb tissues with intramuscular E-selectin gene therapy promotes angiogenesis and reduces tissue loss in a murine hindlimb gangrene model. In this study, we evaluated the effects of E-selectin gene therapy on skeletal muscle recovery, specifically focusing on exercise performance and myofiber regeneration. C57BL/6J mice were treated with intramuscular E-selectin/adeno-associated virus serotype 2/2 gene therapy (E-sel/AAV) or LacZ/AAV2/2 (LacZ/AAV) as control and then subjected to femoral artery coagulation. Recovery of hindlimb perfusion was assessed by laser Doppler perfusion imaging and muscle function by treadmill exhaustion and grip strength testing. After three postoperative weeks, hindlimb muscle was harvested for immunofluorescence analysis. At all postoperative time points, mice treated with E-sel/AAV had improved hindlimb perfusion and exercise capacity. E-sel/AAV gene therapy also increased the coexpression of MyoD and Ki-67 in skeletal muscle progenitors and the proportion of Myh7+ myofibers. Altogether, our findings demonstrate that in addition to improving reperfusion, intramuscular E-sel/AAV gene therapy enhances the regeneration of ischemic skeletal muscle with a corresponding benefit on exercise performance. These results suggest a potential role for E-sel/AAV gene therapy as a nonsurgical adjunct in patients with life-limiting PAD.

A novel autologous cell-based therapy to promote diabetic wound healing.

OBJECTIVES: We have previously shown that stromal cell-derived factor-1α (SDF-1α) is downregulated within diabetic cutaneous wounds, and that direct application of recombinant SDF-1α increases wound closure rates, neovascularization, and endothelial progenitor cell (EPC) recruitment. However, increased wound levels of exogenous SDF-1α results in elevated systemic levels of this proangiogenic chemokine that raises concerns for tumorigenesis and inflammation. We now seek to test the efficacy of a novel, safer cell-based therapy (CBT) employing ex vivo primed bone marrow-derived stem cells (BMDSC) with SDF-1α. We also elucidate the mechanism of action of this new approach for accelerating diabetic wound healing. METHODS: Unfractionated BMDSC from diabetic Lepr mice were incubated for 20 hours with SDF-1α (100 ng/mL) or bovine serum albumin (control). Pretreated BMDSC (1 × 10) were injected subcutaneously into full-thickness skin wounds in Lepr mice (n = 8 per group). Wound closure rates, capillary density, and the recruitment of EPC were assessed with serial photography, DiI perfusion, confocal microscopy, and immunohistochemistry. The expression of molecular targets, which may mediate prohealing/proangiogenic effects of SDF-1α-primed BMDSC was evaluated by polymerase chain reaction array and immunoblotting assay. The biological function of a potential mediator was tested in a mouse wound-healing model. Serum SDF-1α levels were measured with enzyme-linked immunosorbent assay (ELISA). RESULTS: SDF-1α-primed BMDSC significantly promote wound healing (P < 0.0001), neovascularization (P = 0.0028), and EPC recruitment (P = 0.0059). Gene/protein expression studies demonstrate upregulation of Ephrin Receptor B4 and plasminogen as downstream targets potentially mediating the prohealing and proangiogenic responses. Ex vivo BMDSC activation and the subsequent inoculation of cells into wounds does not increase systemic SDF-1α levels. CONCLUSIONS: We report a novel CBT that is highly effective in promoting healing and neovascularization in a murine model of type 2 diabetes. Furthermore, we identify new molecular targets that may be important for advancing the field of wound healing.

A new arteriovenous fistula model to study the development of neointimal hyperplasia.

This study describes an alternative arteriovenous fistula (AVF) model in the rat in which the animals develop significant neointimal hyperplasia (NIH) not only at the distal anastomotic site, but also throughout the fistula body. This aortocaval fistula was established by anastomosing the distal end of the renal vein to the abdominal aorta after unilateral nephrectomy. The increased hemodynamic stress resulting from exposing the renal vein to the arterial circulation induced venous NIH as early as 7 days after surgery. This experimental AVF was characterized by the early lack of endothelium, the accumulation of proliferating vascular smooth muscle cells and the neovascularization of the fistula adventitia. In summary, we have described an informative animal model to study the pathobiology of NIH in native AVF.

Slit-Robo: neuronal guides signal in tumor angiogenesis.

Slit and Roundabout (Robo) are well-characterized for neuron and leukocyte guidance. Their governing roles have now been expanded to control tumor-endothelial cell communication and mediate tumor-induced angiogenesis.