Joel Reichensperger

Prevention of age-related dysregulation of calcium dynamics by estrogen in neurons

To determine the impact of aging and 17beta-estradiol on neuronal Ca2+ homeostasis, intracellular Fura-2 Ca2+-imaging was conducted during 20-pulses of glutamate in hippocampal neurons cultured from embryonic (E18), middle-age (10 months) and old (24 months) rat brain. Marked age-related differences in intracellular Ca2+ ([Ca2+]i) homeostasis and striking regulation by 17beta-estradiol were seen. Embryonic neurons exhibited the greatest capacity to regulate Ca2+ homeostasis followed by middle-age neurons. In old neurons, the first peak [Ca2+]i was substantially greater than at other ages and the return to baseline Ca2+ rapidly dysregulated with an inability to restore [Ca2+]i following the first glutamate pulse which persisted throughout the 20 pulses. 17beta-Estradiol pretreatment of old neurons profoundly attenuated the peak [Ca2+]i rise and delayed the age-associated dysregulation of baseline [Ca2+]i, normalizing responses to those of middle-age neurons treated with estradiol. The efficacy of 17beta-estradiol extended below 10 pg/ml with full protection against toxicity from glutamate and Abeta (1-40). These results demonstrate age-associated dysregulation of [Ca2+]i homeostasis which was largely prevented by 17beta-estradiol with implications for estrogen/hormone therapy.

Stimulation of the follicular bulge LGR5+ and LGR6+ stem cells with the gut-derived human alpha defensin 5 results in decreased bacterial presence, enhanced wound healing, and hair growth from tissues devoid of adnexal structures.

Background: Discovery of leucine-rich repeat-containing G-protein–coupled receptors 5 and 6 (LGR5 and LGR6) as markers of adult epithelial stem cells of the skin and intestine permits researchers to draw on the intrinsic cellular fundamentals of wound healing and proliferation dynamics of epithelial surfaces. In this study, the authors use the intestine-derived human alpha defensin 5 to stimulate epithelial proliferation, bacterial reduction, and hair production in burn wound beds to provide the field with initial insight on augmenting wound healing in tissues devoid of adnexal stem cells. Methods: Murine third-degree burn wound beds were treated with (1) intestine-derived human alpha defensin 5, (2) skin-derived human beta defensin 1, and (3) sulfadiazine to determine their roles in wound healing, bacterial reduction, and hair growth. Results: The human alpha defensin 5 peptide significantly enhanced wound healing and reduced basal bacterial load compared with human beta defensin 1 and sulfadiazine. Human alpha defensin 5 was the only therapy to induce LGR stem cell migration into the wound bed. In addition, gene heat mapping showed significant mRNA up-regulation of key wound healing and Wnt pathway transcripts such as Wnt1 and Wisp1. Ex vivo studies showed enhanced cell migration in human alpha defensin 5–treated wounds compared with controls. Conclusions: Application of human alpha defensin 5 increases LGR stem cell migration into wound beds, leading to enhanced healing, bacterial reduction, and hair production through the augmentation of key Wnt and wound healing transcripts. These findings can be used to derive gut protein–based therapeutics in wound healing.

Transplantation of the LGR6+ epithelial stem cell into full-thickness cutaneous wounds results in enhanced healing, nascent hair follicle development, and augmentation of angiogenic analytes.

Background: The recently discovered leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6+) epithelial stem cell located within the follicular bulge of the adnexal compartment is capable of producing all cellular lineages of the skin. In this study, the authors sought to determine whether these cells can be transplanted for use as a type of cellular therapy for the repair of full-thickness wounds in which the native stem cell niche has been obliterated. Methods: Full-thickness murine skin was harvested and LGR6+GFP epithelial stem cells were isolated using fluorescence-activated cell sorting. This enriched epithelial stem cell population was then transplanted by means of local injection into wound beds on the dorsum of nude mice. Viability, migration, healing, the development of nascent hair follicles, and gene and proteomic expression studies were performed to determine whether the engraftment of LGR6+GFP epithelial stem cells enhanced healing when compared with controls. Results: Wound beds receiving LGR6+GFP epithelial stem cells showed enhanced healing; nascent follicle growth; and augmentation of the Wnt, vascular endothelial growth factor, epidermal growth factor, and platelet-derived growth factor pathways when compared with controls. Conclusions: The LGR6+ epithelial stem cells appear to hold great promise for the development of a clinically useful stem cell–based therapy for the repair of full-thickness wounds and hair regeneration. These results indicate that transplantation of LGR6+ epithelial stem cells promotes epithelialization, hair growth, and angiogenesis in tissues destined for scar formation.

Adipose-derived stem cell to epithelial stem cell transdifferentiation: a mechanism to potentially improve understanding of fat grafting's impact on skin rejuvenation.

BACKGROUND Recent evidence suggests that lipofilling improves overlying skin composition and appearance. Adipose-derived stem cells (ADSC) have been implicated. OBJECTIVE The authors identify ADSC transdifferentiation into epithelial stem cells through coexpression of GFP+ (green fluorescent protein positive) ADSC with the epithelial stem cell marker p63 in an in vivo fat grafting model. METHODS Six male, GFP+ mice served as adipose tissue donors. Twelve nude mice served as recipients. Recipients were subdivided into 2 arms (6 mice/each arm) and received either whole-fat specimen (group 1) or isolated and purified ADSC + peptide hydrogel carrier (group 2) engrafted into a 1-cm(2) left parascapular subdermal plane. The right parascapular subdermal plane served as control. Skin flaps were harvested at 8 weeks and subjected to (1) confocal fluorescent microscopy and (2) reverse transcriptase polymerase chain reaction (RT-PCR) for p63 mRNA expression levels. RESULTS Gross examination of skin flaps demonstrated subjectively increased dermal vessel presence surrounding whole-fat and ADSC specimens. The GFP+ cells were seen within overlying dermal architecture after engraftment and were found to coexpress p63. Significantly increased levels of p63 expression were found in the ADSC + hydrogel skin flaps. CONCLUSIONS We offer suggestive evidence that GFP+ ADSC are found within the dermis 8 weeks after engraftment and coexpress the epithelial stem cell marker p63, indicating that ADSC may transdifferentiate into epithelial stem cells after fat grafting. These findings complement current understanding of how fat grafts may rejuvenate overlying skin.

Reduced basal and lipopolysaccharide-stimulated adenosine A1 receptor expression in the brain of nuclear factor-κB p50−/− mice

Adenosine promotes cytoprotection under conditions of infection, ischemic preconditioning and oxidative stress. Previous studies from our laboratory indicate that the expression of the adenosine A1 receptor (A1AR) is induced by oxidative stress via activation of nuclear factor (NF)-kappaB. The prototypic transcription factor is composed of homo- or heterodimers of p50 and p65 subunits. To determine the role of NF-kappaB in the regulation of the A1AR in vivo, we compared the A1AR RNA and protein levels in the brains of mice lacking the p50 subunit of NF-kappaB (p50-/- mice) and age-matched B6129PF2/J (F2) controls. Radioligand binding assays in the cortex revealed a significantly lower number of A(1)AR (maximal binding capacity, Bmax) in the cortex of p50-/- mice (151+/-62 fmol/mg protein) versus 479+/-181 fmol/mg protein in the F2 (N=5 per strain, P<0.05), but no change in the equilibrium dissociation constant. Similar reductions in A1AR were measured in the hippocampus, brain stem and hypothalamus and in peripheral tissues, such as the adrenal gland, kidney and spleen. Estimation of the A1AR following purification by antibody affinity columns also indicated reduced A1AR in the p50-/- mice cortex, as compared with the F2 mice. A1AR immunocytochemistry indicates distinct neuronal labeling in the F2 cortex, which was substantially reduced in similar sections obtained from p50-/- mice. The p50-/- mice expressed lower levels of A1AR mRNA than F2 mice, as determined by real time PCR. Quantitation of the A1AR transducing G proteins by Western blotting show significantly less Galphai3, no change in Galphai1, but higher levels of Galphao and Gbeta in the cortices of p50-/-, as compared with F2 mice. Administration of bacterial lipopolysaccharide (LPS), an activator of NF-kappaB, increased A1AR expression in the cortices of F2 mice but not p50-/- mice. Cortical neurons cultures prepared from p50-/- mice showed a greater degree of apoptosis, compared with neurons from F2 mice. Activation of the A1AR reduced apoptosis with greater efficacy in cultures from F2 than p50-/- mice. Taken together, these data support a role for NF-kappaB in determining both the basal and LPS-stimulated A1AR expression in vivo which could contribute to neuronal survival.

Early growth response factor-1: expression in a rabbit flexor tendon scar model.

Background: Adhesion formation limits functional recovery after flexor tendon repair. Various growth factors have been implicated in the adhesion scar process. Early growth response factor-1 (EGR-1), a transcription factor associated with synthesis of a variety of key fibrotic growth factors and expression of extracellular matrix genes, has never been identified in a tendon repair model. Methods: Thirty New Zealand White rabbit forepaws underwent laceration and repair of the middle digit flexor digitorum profundus equivalent in zone II. Sodium morrhuate, a topical sclerosing agent, or phosphate-buffered saline, a standard control, was applied to the repair during closure of the tendon sheath. Tendons were harvested from operated and unoperated forepaws at increasing time intervals (1, 3, 7, 14, and 28 days). Tissues were analyzed by immunohistochemistry and Masson trichrome staining. Results: Immunohistochemistry demonstrated that EGR-1 is expressed at the site of tendon repair, along the epitenon of the tendon, and in the infiltrate of inflammatory cells in the surrounding sheath-scar matrix. Control, unoperated tendons demonstrated baseline EGR-1 expression within epitenon cells. EGR-1 was maximally expressed on postoperative day 7. Sodium morrhuate and phosphate-buffered saline demonstrated no difference in their ability to augment tendon adhesion scar formation. Conclusions: Findings demonstrate the following: (1) EGR-1 expression is increased in the tendon wound environment after flexor tendon laceration repair; (2) normal epitenon cells have low, baseline levels of EGR-1 expression; and (3) sodium morrhuate does not augment scar matrix production more than phosphate-buffered saline. The ideal tendon scar model was not generated.

Transplantation of an LGR6+ Epithelial Stem Cell-Enriched Scaffold for Repair of Full-Thickness Soft-Tissue Defects: The In Vitro Development of Polarized Hair-Bearing Skin.

Background: Recent literature has shown that full-thickness wounds, devoid of the stem cell niche, can subsequently be reconstructed with functional skin elements following migration of the LGR6+ epithelial stem cell into the wound bed. In this study, the authors use a variety of LGR6+ epithelial stem cell–seeded scaffolds to determine therapeutic utility and regenerative potential in the immediate reconstruction of full-thickness wounds. Methods: Isolated LGR6+ epithelial stem cells were seeded onto a spectrum of acellular matrices and monitored in both in vitro and in vivo settings to determine their relative capacity to regenerate tissues and heal wounds. Results: Wound beds containing LGR6+ stem cell–seeded scaffolds showed significantly augmented rates of healing, epithelialization, and hair growth compared with controls. Gene and proteomic expression studies indicate that LGR6+ stem cell–seeded constructs up-regulate WNT, epidermal growth factor, and angiogenesis pathways. Finally, the addition of stromal vascular fraction to LGR6+ stem cell–seeded constructs induces polarized tissue formation, nascent hair growth, and angiogenesis within wounds. Conclusions: LGR6+ stem cells are able to undergo proliferation, differentiation, and migration following seeding onto a variety of collagen-based scaffolding. In addition, deployment of these constructs induces epithelialization, hair growth, and angiogenesis within wound beds. The addition of stromal vascular fraction to LGR6+ stem cell–containing scaffolds initiated an early form of tissue polarization, providing for the first time a clinically applicable stem cell–based construct that is capable of the repair of full-thickness wounds and hair regeneration. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Regulation of ADSC Osteoinductive Potential Using Notch Pathway Inhibition and Gene Rescue: A Potential On/Off Switch for Clinical Applications in Bone Formation and Reconstructive Efforts.

Background: Although there has been tremendous research in the ability of mesenchymal-derived adipose derived stem cells (ADSCs) to form bone, less is known regarding the molecular mechanisms that regulate the osteogenic potential of ADSCs. Notch, which consists of a key family of regulatory ligands involved in bone formation, is expressed in the bone marrow–derived mesenchymal stem cell niche and is critical for proliferation, migration, and ultimately osseous differentiation. The authors investigate how Notch impacts ADSC proliferation and osteogenic differentiation to determine a translatable application of these cells in bone regeneration. Methods: Enriched ADSC populations were isolated from tissue and examined for their ability to respond to Notch pathway signaling events. Proliferation, viability, extracellular matrix deposition, and osteoinduction were assessed following Notch activation and inhibition. Notch pathway rescue was conducted using a lentiviral vector encoding a downstream Notch-1 intracellular domain (NICD). Results: Proliferation, osteogenic induction, and the ability to form bone elements were reduced following Notch inhibition (p < 0.05). However, ADSCs, while in the presence of the Notch inhibition, were able to be rescued following lentiviral transduction with NICD, restoring osteogenic potential at both the molecular and cellular functional levels (p < 0.05). Conclusions: These data suggest a potential translatable “on/off switch,” using endogenous Notch signaling to regulate the proliferation, differentiation, and osteogenic potential of ADSCs. Although Notch inhibition reduced ADSC proliferation and down-regulated osteoinduction, targeted gene therapy and the delivery of the downstream NICD peptide restored bone formation, suggesting pragmatic clinical utility of ADSCs for bone regeneration.

A Microbiological and Ultrastructural Comparison of Aseptic versus Sterile Acellular Dermal Matrix as a Reconstructive Material and a Scaffold for Stem Cell Ingrowth

Background: Recent data suggest an increased risk for infection when acellular dermal matrix is used in breast reconstruction. This may be because some acellular dermal matrices are actually not terminally sterilized but are instead “aseptically processed.” This study evaluates aseptic and sterile matrices for evidence of bacterial contamination and whether or not terminal sterilization affects matrix collagen architecture and stem cell ingrowth. Methods: Five separate samples of 14 different matrices were analyzed by fluorescent in situ hybridization using a bacterial DNA probe to detect bacterial DNA on the matrices. Separate samples were incubated for bacteria, acid-fast bacilli, and fungi for 2 to 6 weeks to detect living organisms. The impact of terminal sterilization on the collagen network and stem cell ingrowth on the matrices was then assessed. Results: Traces of bacterial DNA were encountered on all matrices, with more bacteria in the aseptic group compared with the sterile group (3.4 versus 1.6; p = 0.003). The number of positive cultures was the same between groups (3.8 percent). Electron microscopy demonstrated decreased collagen organization in the sterile group. Stem cell seeding on the matrices displayed a wide variation of cellular ingrowth between matrices, with no difference between aseptic and sterile groups (p = 0.2). Conclusions: Although there was more evidence of prior bacterial contamination on aseptically processed matrices compared with sterile matrices; clinical cultures did not differ between groups. Terminal sterilization does not appear to affect stem cell ingrowth but may come at the cost of damaging the collagen network. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Defining the Salvage Time Window for the Use of Ischemic Postconditioning in Skeletal Muscle Ischemia Reperfusion Injury.

BACKGROUND The aim of this study was to determine the optimal salvage time window within which ischemic postconditioning can be used to ameliorate ischemia/reperfusion (I/R) injury in skeletal muscle. METHODS A total of 48 Sprague-Dawley rats were divided into two groups: I/R only (control) and I/R with postconditioning. Subgroups were divided by duration of ischemia (2, 4, 6, and 8 hours). A pedicled gracilis muscle model was used. The postconditioning protocol consisted of six cycles of 15 seconds of reperfusion followed by 15 seconds of ischemia (total time = 3 minutes). Muscles were harvested 24 hours after I/R injury to examine tissue viability, histology, myeloperoxidase activity, and protective gene expression. RESULTS Postconditioning groups showed improved muscle viability after 4 and 6 hours of ischemia time as compared with controls (p < 0.05). Higher expression of mitochondrial complexes I, II, III, endothelial nitric oxide synthase, inducible nitric oxide synthase, and Bcl-2 were observed in the postconditioning group after 4 and 6 hours of ischemia (p < 0.05). Lower expression of tumor necrosis factor-α and caspase 3 was observed in the postconditioning group at 4 hours (p < 0.05). Myeloperoxidase activity was similar in both groups at all-time points except 8 hours ischemia, where the control group had higher activity (p < 0.05). CONCLUSION Results of this study demonstrate that the effective time window within which postconditioning is most effective for the salvage of skeletal muscle is between 4 and 6 hours of ischemia. Postconditioning offered improved mitochondrial and vascular function with decreased inflammation and cell death. This may be clinically useful as a postinjury salvage technique to attenuate I/R injury after 4 to 6 hours of ischemia.